Non-replicative transduction particles and transduction particle-based reporter systems

ABSTRACT

Methods and systems are provided for packaging reporter nucleic acid molecules into non-replicative transduction particles for use as reporter molecules. The non-replicative transduction particles can be constructed from viruses and use viral transduction and replication systems. The reporter nucleic acid molecules include a reporter gene, such as a reporter molecule or selectable marker, for detecting target genes or cells. Methods and systems are provided for detection of cells and target nucleic acid molecules using the non-replicative transduction particles as reporter molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/183,303, filed on Jun. 15, 2016 which is a continuation of U.S.patent application Ser. No. 14/550,335, filed on Nov. 21, 2014, now U.S.Pat. No. 9,388,453, which claims the benefit of priority toInternational Application No. PCT/US2014/026536, filed on Mar. 13, 2014,U.S. Provisional Application No. 61/779,177, filed on Mar. 13, 2013,U.S. Provisional Application No. 61/897,040, filed on Oct. 29, 2013, andU.S. Provisional Application No. 61/939,126, filed on Feb. 12, 2014,each of which is hereby incorporated in its entirety by reference.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronictext file named “33165—US5.txt”, having a size in bytes of 65 kb, andcreated on Jan. 5, 2017. The information contained in this electronicfile is hereby incorporated by reference in its entirety pursuant to 37CFR § 1.52(e)(5).

BACKGROUND OF THE INVENTION Field of the Invention

The invention relates to methods and compositions for packaging anddelivery of non-replicative transduction reporter molecules into cellsfor detecting target genes in cells.

Description of the Related Art

A transduction particle refers to a virus capable of delivering anon-viral nucleic acid into a cell. Viral-based reporter systems havebeen used to detect the presence of cells and rely on the lysogenicphase of the virus to allow expression of a reporter molecule from thecell. These viral-based reporter systems use replication-competenttransduction particles that express reporter molecules and cause atarget cell to emit a detectable signal.

However, the lytic cycle of the virus has been shown to be deleteriousto viral-based reporter assays. Carriere, C. et al., Conditionallyreplicating luciferase reporter phages: Improved sensitivity for rapiddetection and assessment of drug susceptibility of Mycobacteriumtuberculosis. Journal of Clinical Microbiology, 1997. 35(12): p.3232-3239. Carrière et al. developed M. tuberculosis/bacillusCalmette-Guèrin (BCG) luciferase reporter phages that have their lyticcycles suppressed at 30° C., but active at 37° C. Using this system,Carrière et al. have demonstrated the detection of BCG using phagereporters with a suppressed lytic cycle.

There are disadvantages, however, associated with suppressing but noteliminating the replication functions of the bacteriophage inbacteriophage-based reporter assays. First, controlling replicationfunctions of the bacteriophage imposes limiting assay conditions. Forexample, the lytic cycle of the reporter phage phAE40 used by Carrièreet al. was repressed when the phage was used to infect cells at thenon-permissive temperature of 30° C. This temperature requirementimposed limiting conditions on the reporter assay in that the optimumtemperature for the target bacteria was 37° C. These limiting conditionshinder optimum assay performance.

Moreover, the replication functions of the virus are difficult tocontrol. The replication of the virus should be suppressed during theuse of the transduction particles as a reporter system. For example, thelytic activity of the reporter phage phAE40 reported by Carrière et al.was reduced but was not eliminated, resulting in a drop in luciferasesignal in the assay. Carrière et al. highlighted possible causes for theresulting drop in reporter signal, such as intact phage-expressed genesand temperature limitations of the assay, all stemming from the factthat the lytic cycle of the phage reporter was not eliminated.

Reporter assays relying on the natural lysogenic cycle of phages can beexpected to exhibit lytic activity sporadically. In addition, assaysthat rely on the lysogenic cycle of the phage can be prone tosuperinfection immunity from target cells already lysogenized with asimilar phage, as well as naturally occurring host restriction systemsthat target incoming virus nucleic acid, thus limiting the host range ofthese reporter phages.

In other examples, transduction particle production systems are designedto package exogenous nucleic acid molecules, but the transductionparticle often contains a combination of exogenous nucleic acidmolecules and native progeny virus nucleic acid molecules. The nativevirus can exhibit lytic activity that is a hindrance to assayperformance, and the lytic activity of the virus must be eliminated inorder to purify transduction particles. However, this purification isgenerally not possible. In U.S. 2009/0155768 A, entitled ReporterPlasmid Packaging System for Detection of Bacteria, Scholl et al.describes the development of such a transduction particle system. Theproduct of the system is a combination of reporter transductionparticles and native bacteriophage (FIG. 8 in the reference). Althoughthe authors indicate that the transduction particle and nativebacteriophage can be separated by ultracentrifugation, this separationis only possible in a system where the transduction particle and thenative virus exhibit different densities that would allow separation byultracentrifugation. While this characteristic is exhibited by thebacteriophage T7-based packaging system described in the reference, thisis not a characteristic that is generally applicable for other virussystems. It is common for viral packaging machinery to exhibit headfulpackaging that would result in native virus and transduction particlesto exhibit indistinguishable densities that cannot be separated byultracentrifugation. Virus packaging systems also rely on a minimumamount of packaging as a requirement for proper virus structuralassembly that results in native virus and transduction particles withindistinguishable densities.

Thus, there is a need for non-replicative transduction particles that donot suffer from the deleterious effects from lytic functions of thevirus and the possibility of being limited by superinfection immunityand host restriction mechanisms that target virus nucleic acid moleculesand viral functions, all of which can limit the performance of thereporter assay by increasing limits of detection and resulting in falsenegative results.

Even where transduction particles have been engineered, methods forusing the transduction particles to detect and report the presence oftarget nucleic acid molecules in cells have limitations. Some methodsrequire disruption of the cell and cumbersome techniques to isolate anddetect transcripts in the lysate. Detection methods include usinglabeled probes such as antibodies, aptamers, or nucleic acid probes.Labeled probes directed to a target gene can result in non-specificbinding to unintended targets or generate signals that have a highsignal-to-noise ratio. Therefore, there is a need for specific,effective and accurate methods for detection and reporting of endogenousnucleic acid molecules in cells.

Accordingly, methods and systems are needed for generatingnon-replicative transduction particles that allow packaging andexpression of reporter molecules in cells, while eliminatingreplication-competent progeny virus. Effective and accurate methods fordetecting molecules in cells using the expressed reporter molecules arealso needed.

SUMMARY OF THE INVENTION

Disclosed herein is a bacterial cell packaging system for packaging areporter nucleic acid molecule into a non-replicative transductionparticle, said bacterial cell comprising a lysogenized bacteriophagegenome lacking a bacteriophage gene encoding a packaging initiation sitesequence, wherein deletion of said bacteriophage gene prevents packagingof a bacteriophage nucleic acid molecule into said non-replicativetransduction particle; and a reporter nucleic acid molecule comprising asecond bacteriophage gene, wherein said second bacteriophage geneencodes a packaging initiation site sequence and facilitates thepackaging a replica of said reporter nucleic acid molecule into saidnon-replicative transduction particle, wherein said second bacteriophagegene is capable of expressing a protein that is encoded by said gene,wherein said replica of said reporter nucleic acid molecule forms areplicon amenable to packaging into said non-replicative transductionparticle.

In some embodiments, the reporter nucleic acid molecule is operativelylinked to a promoter. In another embodiment, the promoter is selectedfor contributing to reactivity of a reporter molecule expressed fromsaid reporter nucleic acid molecule in said bacterial cell. In oneembodiment, the reporter nucleic acid molecule comprises an origin ofreplication. In yet another embodiment, the replicon comprises aconcatamer amenable to packaging into said non-replicative transductionparticle.

In an embodiment, the first and said second bacteriophage genes eachcomprises a pacA gene of the Enterobacteriaceae bacteriophage P1 andcomprises said packaging initiation site sequence. In one embodiment,the second bacteriophage gene comprises the sequence of SEQ ID NO:9. Inanother embodiment, the replicon is the Enterobacteriaceae bacteriophageP1 lytic replicon. In certain embodiments, the replicon comprises a C1repressor-controlled P53 promoter, a promoter P53 antisense, a repLgene, and an in-frame deletion of a kilA gene. In one embodiment, thereplicon comprises of the sequence of SEQ ID NO:3.

In yet another embodiment, the first and said second bacteriophage geneseach comprises a small terminase (terS) gene comprising said packaginginitiation site sequence. In one embodiment, the terS gene is a S.aureus bacteriophage φ11 or φ80α terS gene.

In another embodiment, the replicon is derived from a S. aureus pT181plasmid origin of replication. In yet another embodiment, the repliconcomprises the sequence of SEQ ID NO:5. In some embodiments, thepackaging initiation site sequence of said second bacteriophage genecomprises a pac-site. In other embodiments, the pac-site of said secondbacteriophage gene comprises the sequence of SEQ ID NO:7. In one aspect,the packaging initiation site sequence of said second bacteriophage genecomprises a cos-site. In another aspect, the packaging initiation sitesequence of said second bacteriophage gene comprises a concatamerjunction.

In another aspect, a plasmid comprises said reporter nucleic acidmolecule. In one aspect, the second bacteriophage gene is operativelylinked to a promoter. In another embodiment, the promoter is aninducible promoter or a constitutive promoter. In one embodiment, thebacteriophage comprises the Enterobacteriaceae bacteriophage P1. In yetanother embodiment, the bacteriophage comprises a S. aureusbacteriophage φ80α or a bacteriophage φ11. In one aspect, the bacterialcell comprises an E. coli cell. In another aspect, the bacterial cellcomprises an S. aureus cell. In yet another embodiment, the bacterialcell comprises a Gram-negative cell. In other embodiments, the bacterialcell comprises a Gram-positive cell.

In another aspect, the reporter nucleic acid molecule comprises areporter gene. In one aspect, the reporter gene encodes a detectableand/or a selectable marker. In certain aspects, the reporter gene isselected from the group consisting of enzymes mediating luminescencereactions (luxA, luxB, luxAB, luc, mc, nluc), enzymes mediatingcolorimetric reactions (lacZ, HRP), fluorescent proteins (GFP, eGFP,YFP, RFP, CFP, BFP, mCherry, near-infrared fluorescent proteins),affinity peptides (His-tag, 3X-FLAG), and selectable markers (ampC,tet(M), CAT, erm). In another aspect, the reporter nucleic acid moleculecomprises an aptamer. In yet another aspect, the reporter nucleic acidmolecule comprises a nucleic acid transcript sequence that iscomplementary to a second sequence in said reporter nucleic acidmolecule.

In one embodiment, the nucleic acid transcript sequence is complementaryto a cellular transcript. In another embodiment, the nucleic acidtranscript sequence comprises a cis-repressing sequence. In yet anotherembodiment, the replica of said reporter nucleic acid molecule comprisesa nucleic acid transcript sequence that is complementary to a secondsequence in said replica of said reporter nucleic acid molecule, whereinthe nucleic acid transcript sequence is complementary to a cellulartranscript and wherein said nucleic acid transcript sequence comprises acis-repressing sequence.

In some embodiments, the method for packaging a reporter nucleic acidmolecule into a non-replicative transduction particle, comprisingproviding conditions to said bacterial cell described herein that inducea lytic phase of said bacteriophage to produce non-replicativetransduction particles packaged with said reporter nucleic acidmolecule; and isolating said non-replicative transduction particlecomprising said reporter nucleic acid molecule. In one embodiment, thenon-replicative transduction particle does not contain a replicatedbacteriophage genome. In another embodiment, induction of said lyticphase triggers excision of said genomic island nucleic acid moleculefrom said genome of said bacterial cell.

In another embodiment, the composition comprising said non-replicativetransduction particle comprising a replica of said reporter nucleic acidmolecule produced from the method described herein.

The invention comprises a bacterial cell packaging system for packaginga reporter nucleic acid molecule into a non-replicative transductionparticle, said bacterial cell comprising a lysogenized bacteriophagegenome comprising a first bacteriophage packaging initiation sitesequence, wherein said first bacteriophage packaging initiation sitesequence comprises a mutation that prevents packaging of a bacteriophagenucleic acid molecule into said non-replicative transduction particle;and a reporter nucleic acid molecule comprising a second bacteriophagepackaging initiation site sequence, wherein said second bacteriophagepackaging initiation site sequence lacks said mutation and facilitatesthe packaging of a replica of said reporter nucleic acid molecule intosaid non-replicative transduction particle, wherein said replica of saidreporter nucleic acid molecule forms a replicon for packaging into saidnon-replicative transduction particle.

In one embodiment, the reporter nucleic acid molecule is operativelylinked to a promoter. In another embodiment, the promoter is selectedfor contributing to reactivity of a reporter molecule expressed fromsaid reporter nucleic acid molecule in said bacterial cell. In yetanother embodiment, the reporter nucleic acid molecule comprises anorigin of replication. In one embodiment, the replicon comprises aconcatamer amenable to packaging into said non-replicative transductionparticle. In another aspect, the first and said second bacteriophagepackaging initiation site sequences each comprise a packaging initiationsite sequence from a small terminase gene. In one aspect, the first andsaid second bacteriophage packaging initiation site sequences eachcomprise a pac-site sequence from a pacA gene of the Enterobacteriaceaebacteriophage P1. In another aspect, the first bacteriophage packaginginitiation site sequence comprises SEQ ID NO:2. In yet another aspect,the second bacteriophage packaging initiation site sequence comprisesSEQ ID NO:1. In one embodiment, the replicon comprises anEnterobacteriaceae bacteriophage P1 lytic replicon. In anotherembodiment, the replicon comprises a C1 repressor-controlled P53promoter, a promoter P53 antisense, a repL gene, and an in-framedeletion of a kilA gene. In another aspect, the replicon comprises thesequence of SEQ ID NO:3. In certain aspects, the first and said secondbacteriophage packaging initiation site sequences each comprise apac-site sequence from a small terminase (terS) gene of an S. aureusbacteriophage φ11 or φ80α. In another aspect, the replicon is derivedfrom a S. aureus pT181 plasmid origin of replication. In yet anotheraspect, the replicon comprises the sequence of SEQ ID NO:5. In oneaspect, the first bacteriophage packaging initiation site sequencecomprises the sequence of SEQ ID NO:2. In some embodiments, the secondbacteriophage packaging initiation site sequence comprises the sequenceof SEQ ID NO:1. In other embodiments, the packaging initiation sitesequence comprises a pac-site. In another embodiment, the packaginginitiation site sequence comprises a cos-site. In yet anotherembodiment, the packaging initiation site sequence comprises aconcatamer junction. In some embodiments, the mutation in said firstbacteriophage packaging initiation site sequence comprises a silentmutation. In another embodiment, the mutation in said firstbacteriophage packaging initiation site sequence prevents cleavage ofsaid packaging initiation sequence. In another embodiment, a plasmidcomprises said reporter nucleic acid molecule. In one embodiment, thebacteriophage comprises Enterobacteriaceae bacteriophage P1.

In another embodiment, the bacteriophage comprises the S. aureusbacteriophage φ11 or φ80α. In one embodiment, the bacterial cellcomprises an E. coli cell. In another embodiment, the bacterial cellcomprises an S. aureus cell. In some embodiments, the bacterial cellcomprises a Gram-negative bacterial cell. In one aspect, the bacterialcell comprises a Gram-positive bacterial cell. In another aspect, thereporter nucleic acid molecule comprises a reporter gene. In yet anotheraspect, the reporter gene encodes a detectable marker and/or aselectable marker.

In other aspects, the reporter gene is selected from the groupconsisting of: genes encoding enzymes mediating luminescence reactions(luxA, luxB, luxAB, luc, mc, nluc), genes encoding enzymes mediatingcolorimetric reactions (lacZ, HRP), genes encoding fluorescent proteins(GFP, eGFP, YFP, RFP, CFP, BFP, mCherry, near-infrared fluorescentproteins), nucleic acid molecules encoding affinity peptides (His-tag,3X-FLAG), and genes encoding selectable markers (ampC, tet(M), CAT,erm). In another aspect, the reporter nucleic acid molecule comprises anaptamer. In other aspects, the replicon is packaged into saidnon-replicative transduction particle by bacteriophage packagingmachinery. In some embodiments, the reporter nucleic acid moleculecomprises a nucleic acid transcript sequence that is complementary to asecond sequence in said reporter nucleic acid molecule. In anotherembodiment, the nucleic acid transcript sequence is complementary to acellular transcript.

In one aspect, the nucleic acid transcript sequence comprises acis-repressing sequence. In another aspect, the replica of said reporternucleic acid molecule comprises a nucleic acid transcript sequence thatis complementary to a second sequence in said replica of said reporternucleic acid molecule, wherein said nucleic acid transcript sequence iscomplementary to a cellular transcript, and wherein said nucleic acidtranscript sequence comprises a cis-repressing sequence.

In certain aspects, the method for packaging a reporter nucleic acidmolecule into a non-replicative transduction particle, comprising:providing conditions to said bacterial cell described herein that inducea lytic phase of said bacteriophage to produce non-replicativetransduction particles packaged with said reporter nucleic acidmolecule; and isolating said non-replicative transduction particlecomprising said reporter nucleic acid molecule.

In other aspects, the non-replicative transduction particle does notcontain a replicated bacteriophage genome. In one aspect, the inductionof said lytic phase triggers excision of said genomic island nucleicacid molecule from said genome of said bacterial cell.

In another aspect, the invention comprises a composition comprising saidnon-replicative transduction particle comprising a replica of saidreporter nucleic acid molecule produced from said method describedherein.

In one aspect, the invention includes a bacterial cell packaging systemfor packaging a reporter nucleic acid molecule into a non-replicativetransduction particle, said bacterial cell comprising: a lysogenizedbacteriophage genome comprising a first bacteriophage gene comprising adeletion of a packaging initiation site sequence of said firstbacteriophage gene that prevents packaging of a bacteriophage nucleicacid molecule into said non-replicative transduction particle; and areporter nucleic acid molecule comprising a second bacteriophage genecomprising a second packaging initiation site sequence that facilitatesthe packaging a replica of said reporter nucleic acid molecule into saidnon-replicative transduction particle, wherein said second bacteriophagegene encodes a protein, wherein said replica of said reporter nucleicacid molecule forms a replicon for packaging into said non-replicativetransduction particle.

In another aspect, the reporter nucleic acid molecule is operativelylinked to a promoter. In one aspect, the promoter is selected forcontributing to reactivity of a reporter molecule expressed from saidreporter nucleic acid molecule in said bacterial cell. In certainaspects, the reporter nucleic acid comprises an origin of replication.In another aspect, the replicon comprises a concatamer amenable topackaging into said non-replicative transduction particle. In oneaspect, the first and said second bacteriophage genes each comprises apacA gene of the Enterobacteriaceae bacteriophage P1 and comprise saidpackaging initiation site sequence. In another aspect, the firstbacteriophage gene comprises the sequence of SEQ ID NO:6. In certainaspects, the second bacteriophage gene comprises the sequence SEQ IDNO:7. In one aspect, the replicon comprises an Enterobacteriaceaebacteriophage P1 lytic replicon. In yet another aspect, the repliconcomprises a C1 repressor-controlled P53 promoter, a promoter P53antisense, a repL gene, and an in-frame deletion of a kilA gene. Inanother aspect, the replicon comprises the sequence of SEQ ID NO:3. Inother aspects, the first and said second bacteriophage genes eachcomprises a small terminase (terS) gene comprising said packaginginitiation site sequence. In one aspect, the terS gene is a S. aureusbacteriophage φ11 or φ80α terS gene. In another aspect, the firstbacteriophage gene comprises the sequence of SEQ ID NO:8. In yet anotheraspect, the second bacteriophage gene comprises the sequence of SEQ IDNO:9. In one aspect, the replicon is derived from a S. aureus pT181plasmid origin of replication. In one embodiment, the replicon comprisesthe sequence of SEQ ID NO:5, In another embodiment, the packaginginitiation site sequence of said second bacteriophage gene comprises apac-site. In yet another embodiment, the packaging initiation sitesequence of said second bacteriophage gene comprises a cos-site.

In certain embodiments, the packaging initiation site sequence of saidsecond bacteriophage gene comprises a concatamer junction. In oneembodiment, a plasmid comprises said reporter nucleic acid molecule. Inanother embodiment, the second bacteriophage gene is operatively linkedto a promoter. In yet another embodiment, the promoter is an induciblepromoter or a constitutive promoter. In certain embodiments, thebacteriophage comprises the Enterobacteriaceae bacteriophage P1. In oneembodiment, the bacteriophage comprises the S. aureus bacteriophage φ80αor bacteriophage φ11. In other embodiments, the bacterial cell comprisesan E. coli cell. In another embodiment, the bacterial cell comprises anS. aureus cell. In one embodiment, the bacterial cell comprises aGram-negative cell. In another embodiment, the bacterial cell comprisesa Gram-positive cell.

In another aspect, the reporter nucleic acid molecule comprises areporter gene. In one aspect, the reporter gene encodes a detectableand/or a selectable marker. In another aspect, the reporter gene isselected from the group consisting of genes encoding enzymes mediatingluminescence reactions (luxA, luxB, luxAB, luc, ruc, nluc), genesencoding enzymes mediating colorimetric reactions (lacZ, HRP), genesencoding fluorescent proteins (GFP, eGFP, YFP, RFP, CFP, BFP, mCherry,near-infrared fluorescent proteins), nucleic acid molecules encodingaffinity peptides (His-tag, 3X-FLAG), and genes encoding selectablemarkers (ampC, tet(M), CAT, erm). In one embodiment, the reporternucleic acid molecule comprises an aptamer. In another embodiment, thereplicon is packaged into said non-replicative transduction particle bybacteriophage packaging machinery. In yet another embodiment, thereporter nucleic acid molecule comprises a nucleic acid transcriptsequence that is complementary to a second sequence in said reporternucleic acid molecule. In one embodiment, the nucleic acid transcriptsequence is complementary to a cellular transcript. In anotherembodiment, the nucleic acid transcript sequence comprises acis-repressing sequence. In certain embodiments, the replica of saidreporter nucleic acid molecule comprises a nucleic acid transcriptsequence that is complementary to a second sequence in said replica ofsaid reporter nucleic acid molecule, wherein said nucleic acidtranscript sequence is complementary to a cellular transcript andwherein said nucleic acid transcript sequence comprises a cis-repressingsequence.

The invention includes a method for packaging a reporter nucleic acidmolecule into a non-replicative transduction particle, comprising:providing conditions to said bacterial cell that induce a lytic phase ofsaid bacteriophage to produce non-replicative transduction particlespackaged with said reporter nucleic acid molecule; and isolating saidnon-replicative transduction particle comprising said reporter nucleicacid molecule. In one embodiment, the non-replicative transductionparticle does not contain a replicated bacteriophage genome. In anotherembodiment, the induction of said lytic phase triggers excision of saidgenomic island nucleic acid molecule from said genome of said bacterialcell.

In some aspects, the invention includes a composition comprising saidnon-replicative transduction particle comprising a replica of saidreporter nucleic acid molecule produced from said method describedherein.

In another aspect, the invention includes a bacterial cell packagingsystem for packaging a reporter nucleic acid molecule into anon-replicative transduction particle, said bacterial cell comprising: alysogenized bacteriophage genome lacking a packaging gene and comprisinggenes that encode proteins that form said non-replicative transductionparticle; and a genomic island nucleic acid molecule comprising areporter nucleic acid molecule and a packaging gene. In one aspect, thepackaging gene comprises a small terminase (terS) gene. terS genecomprises a S. aureus bacteriophage φ80α terS gene or a bacteriophageφ11 terS gene.

In one aspect, the terS gene comprises the sequence of SEQ ID NO:9. Inanother aspect, the genomic island nucleic acid molecule comprises aSaPIbov2 genomic island nucleic acid molecule. In yet another aspect,the genomic island nucleic acid molecule is selected from the groupconsisting of a SaPI, a SaPI1, a SaPI2, a SaPIbov1 and a SaPibov2genomic island nucleic acid molecule. In another embodiment, thereporter nucleic acid molecule is operatively linked to a promoter. Inyet another embodiment, the reporter nucleic acid molecule comprises anorigin of replication. In some embodiments, the bacteriophage comprisesa S. aureus bacteriophage φ80α or bacteriophage φ11. In otherembodiments, the bacterial cell comprises an S. aureus cell. In oneembodiment, the genomic island nucleic acid molecule comprises anintegrase gene and wherein said integrase gene encodes an integraseprotein for excising and integrating said genomic island nucleic acidmolecule out of and into a bacterial genome of said bacterial cell. Inanother embodiment, the integrase gene comprises the sequence of SEQ IDNO:10. In yet another embodiment, the genomic island nucleic acidmolecule is integrated into a bacterial genome of said bacterial cell.

In certain aspects, the genomic island nucleic acid molecule can bereplicated and forms molecule replicon that is amenable to packaging bythe bacteriophage packaging machinery in said bacterial cell. In anotheraspect, the nucleic acid molecule forms a concatamer. In yet anotheraspect, the replicated genomic island nucleic acid molecule is capableof being packaged into said non-replicative transduction particle. Incertain aspects, the packaging gene comprises a pac site sequence. Inanother aspect, the packaging gene comprises a cos-site sequence. In yetanother embodiment, the packaging gene comprises a concatamer junction.

In other embodiments, the reporter nucleic acid molecule comprises areporter gene. In some embodiments, the reporter gene encodes aselectable marker and/or a selectable marker. In another embodiment, thereporter gene is selected from the group consisting of enzymes mediatingluminescence reactions (luxA, luxB, luxAB, luc, ruc, nluc), enzymesmediating colorimetric reactions (lacZ, HRP), fluorescent proteins (GFP,eGFP, YFP, RFP, CFP, BFP, mCherry, near-infrared fluorescent proteins),affinity peptides (His-tag, 3X-FLAG), and selectable markers (ampC,tet(M), CAT, erm). In certain embodiments, the reporter nucleic acidmolecule comprises an aptamer. In other embodiments, the genomic islandnucleic acid molecule lacks an integrase gene. In another embodiment,the invention includes a bacterial gene comprising an integrase geneoperatively linked to a promoter and wherein said integrase gene encodesan integrase protein for excising and integrating said genomic islandnucleic acid molecule out of and into a bacterial genome of saidbacterial cell. In one embodiment, the reporter nucleic acid moleculecomprises a nucleic acid transcript sequence that is complementary to asecond sequence in said reporter nucleic acid molecule. In otherembodiments, the nucleic acid transcript sequence is complementary to acellular transcript. In yet other embodiments, the nucleic acidtranscript sequence comprises a cis-repressing sequence. In anotherembodiment, the replica of said reporter nucleic acid molecule comprisesa nucleic acid transcript sequence that is complementary to a secondsequence in said replica of said reporter nucleic acid molecule. Inother embodiments, the nucleic acid transcript sequence is complementaryto a cellular transcript. In other embodiments, the nucleic acidtranscript sequence comprises a cis-repressing sequence.

The invention includes a method for packaging a reporter nucleic acidmolecule into a non-replicative transduction particle, comprising:providing conditions to said bacterial cell that induce a lytic phase ofsaid bacteriophage to produce non-replicative transduction particlespackaged with said reporter nucleic acid molecule; and isolating saidnon-replicative transduction particle comprising said reporter nucleicacid molecule. In some embodiments, the non-replicative transductionparticle does not contain a replicated bacteriophage genome. In oneembodiment, the induction of said lytic phase triggers excision of saidgenomic island nucleic acid molecule from said genome of said bacterialcell.

In another embodiment, the invention includes a composition comprisingsaid non-replicative transduction particle comprising a replica of saidreporter nucleic acid molecule produced from said method describedherein.

The invention also includes a method for detecting a presence or anabsence of a bacterial cell in a sample, comprising: introducing into asample a non-replicative transduction particle comprising a reportergene encoding a reporter molecule and lacking a bacteriophage genomeunder conditions such that said non-replicative transduction particlecan transduce said bacterial cell and wherein said reporter gene can beexpressed in said bacterial cell; providing conditions for activation ofsaid reporter molecule; and detecting for a presence or an absence of areporter signal transmitted from said expressed reporter molecule,wherein a presence of said reporter signal correctly indicates saidpresence of said bacterial cell.

In one embodiment, the method achieves at least 80% specificity ofdetection with reference to a standard, at least 90% specificity ofdetection with reference to a standard, or at least 95% specificity ofdetection with reference to a standard. In another embodiment, themethod achieves at least 80% sensitivity of detection with reference toa standard, at least 85% sensitivity of detection with reference to astandard, or at least 90% sensitivity of detection with reference to astandard, or at least 95% sensitivity of detection with reference to astandard. In yet another embodiment, the method achieves at least 95%specificity of detection and at least 90% sensitivity of detection withreference to a standard. In another embodiment, the standard is a Goldstandard. In yet another embodiment, the bacterial cell comprises aMethicillin Resistant Staphylococcus aureus (MRSA) cell. In otherembodiments, the bacterial cell comprises a Methicillin SensitiveStaphylococcus aureus (MSSA) cell.

In another embodiment, the reporter gene encodes a detectable orselectable marker. In one embodiment, the reporter gene is selected fromthe group consisting of genes encoding enzymes mediating luminescencereactions (luxA, luxB, luxAB, luc, ruc, nluc), genes encoding enzymesmediating colorimetric reactions (lacZ, HRP), genes encoding fluorescentproteins (GFP, eGFP, YFP, RFP, CFP, BFP, mCherry, near-infraredfluorescent proteins), nucleic acid molecules encoding affinity peptides(His-tag, 3X-FLAG), and genes encoding selectable markers (ampC, tet(M),CAT, erm). In one embodiment, the reporter gene is operatively linked toa constitutive promoter.

In another aspect, the reporter signal can be detected from a sample ata limit of detection (LoD) of less than 1,000 colony forming units(CFU). In other aspects, the reporter signal can be detected from asample at a limit of detection (LoD) of less than 100 colony formingunits (CFU). In one aspect, the reporter signal can be detected from asample at a limit of detection (LoD) of less than 10 colony formingunits (CFU). In other aspects, the reporter signal can be detected froma sample at a LoD less than five CFU. In another aspect, the reportersignal can be detected from a sample at a LoD of three or less CFU.

In one embodiment, the method includes providing an antibiotic to saidsample at a pre-determined concentration and detecting a presence orabsence of said reporter signal to determine whether said bacterial cellis resistant or sensitive to said antibiotic. In another embodiment, themethod includes providing varying pre-determined concentrationsantibiotic to said sample and detecting the amount of said reportersignal to determine the minimum inhibitory concentration of saidbacterial cell to said antibiotic.

In one aspect, the invention includes a composition comprising a nucleicacid construct that encodes a nucleic acid reporter transcript that iscapable of forming at least two conformations comprising a firstconformation that prevents reporter expression comprising anintramolecular double stranded region comprising a first subsequence anda second subsequence, and a second conformation that lacks saidintramolecular double-stranded region and allows reporter geneexpression, wherein conversion between said first and secondconformations is mediated by competitive binding of a cellulartranscript to said first and/or said second subsequence.

In another aspect, the invention includes a non-replicative transductionparticle comprising said nucleic acid construct. In yet another aspect,the competitive binding of said cellular transcript to said first and/orsaid second subsequence results in said second conformation of saidnucleic acid reporter construct. In one aspect, the first subsequence orsaid second subsequence comprises a cis-repressing sequence. In anotheraspect, the cis-repressing sequence comprises a sequence that iscomplementary or substantially complementary to a portion of saidcellular transcript. In other aspects, the first subsequence or saidsecond subsequence comprises a reporter gene sequence. In yet anotheraspect, the reporter gene sequence comprises a ribosome binding site. Inother aspects, the reporter gene sequence encodes a detectable molecule.In another aspect, the detectable marker comprises a fluorescentmolecule or an enzyme capable of mediating a luminescence orcolorimetric reaction. In one embodiment, the reporter gene sequenceencodes a selectable marker. In another embodiment, the selectablemarker comprises an antibiotic resistance gene.

In other embodiments, the first subsequence and said second subsequenceare located cis to each other on said nucleic acid construct to formsaid intramolecular double stranded region. In certain embodiments, thefirst subsequence and said second subsequence are complementary orsubstantially complementary to each other to form said intramoleculardouble stranded region. In one embodiment, the first subsequence or saidsecond subsequence of said first conformation comprises atranscriptional enhancer sequence, and wherein said transcriptionalenhancer sequence is upstream from a coding region of said reporter genesequence. In another embodiment, the first conformation of said nucleicacid reporter transcript is capable of binding to a cleaving enzyme. Inother embodiments, the first conformation of said nucleic acid reportertranscript is a target for degradation by a cellular enzyme. In otheraspects, the first conformation comprises a non-binding intramolecularregion. In another aspect, the non-binding intramolecular region islocated 3′ of said first subsequence and 5′ of said second subsequence.In other aspects, the non-binding intramolecular region comprises asequence YUNR, wherein Y is a pyrimidine, U is a Uracil, N is anynucleotide, and R is a purine.

In one embodiment, the first subsequence or said second subsequencecomprises a modified sequence of said cellular transcript. In anotherembodiment, the modified sequence comprises a nucleotide substitution.In yet another embodiment, the modified sequence comprises a sequenceinsertion, a deletion or an inversion of said cellular transcript.

The method includes a composition comprising a nucleic acid constructthat encodes a nucleic acid reporter transcript comprising a genereporter sequence and that is capable of forming at least twoconformations of said nucleic acid reporter transcript, a first unstableconformation that prevents translation of said reporter gene sequence insaid nucleic acid reporter transcript, and a second stable conformationresulting from binding of said first unstable conformation with acellular transcript, said second stable secondary conformation allowingtranslation of said reporter gene sequence of said nucleic acid reportertranscript.

In one embodiment, the composition comprises a non-replicativetransduction particle comprising said nucleic acid construct. In anotherembodiment, the cellular transcript binds at a 3′UTR sequence of saidnucleic acid reporter transcript. In one embodiment, the second stablesecondary conformation is formed by cleavage of a portion of a sequenceof said first unstable secondary conformation. In another embodiment,the reporter gene sequence encodes a detectable molecule. In someembodiments, the detectable marker comprises a fluorescent molecule oran enzyme capable of mediating a luminescence or colorimetric reaction.In other embodiments, the reporter gene sequence encodes a selectablemarker. In another embodiment, the selectable marker comprises anantibiotic resistance gene.

The invention also includes a composition comprising a nucleic acidconstruct that encodes a nucleic acid reporter transcript comprising areporter gene sequence and that is capable of forming at least twoconformations of said nucleic acid reporter transcript, comprising afirst conformation that prevents further transcription of said nucleicacid construct, and a second conformation formed upon binding of saidfirst conformation with a cellular transcript, wherein said secondconformation allows transcription of said nucleic acid construct. Insome embodiments, the composition comprises a non-replicativetransduction particle comprising said nucleic acid construct. In anotherembodiment, the nucleic acid reporter transcript comprises acis-repressing sequence.

In one embodiment, the nucleic acid reporter transcript comprises areporter gene sequence. In another embodiment, the first conformationforms from a binding of said cis-repressing sequence to said reportergene sequence. In some embodiments, the first conformation is asubstrate for a cleaving enzyme. In one embodiment, the firstconformation of said nucleic acid reporter transcript comprises asequence that forms a transcription termination structure. In otherembodiments, the binding of said cellular transcript to said sequencethat forms a transcription termination structure results in cleavage ofa portion of said nucleic acid reporter transcript and formation of saidsecond conformation.

The invention comprises a vector comprising a regulatory sequenceoperably linked to a nucleic acid sequence that encodes said nucleicacid reporter transcript described herein.

The invention includes a method for detecting a target transcript in acell, comprising: introducing into said cell said nucleic acid reporterconstruct described herein; and detecting the presence or absence of anoutput signal from said cell, wherein said presence of said outputsignal indicates the presence of the target transcript in said cell. Themethod includes detecting a presence of a bacterial cell based ondetecting said presence of said target transcript.

In one embodiment, the method for detecting a presence of a bacterialcell in a sample comprising introducing into said sample said nucleicacid reporter construct described herein; and detecting the presence orabsence of an output signal from said sample, wherein said presence ofsaid output signal indicates the presence of the bacterial cell in saidsample.

The invention comprises a kit, comprising a compartment for holding asample comprising a cell and said nucleic acid reporter constructdescribed herein; and instructions for detecting the presence or absenceof an output signal from said sample, wherein the presence of the outputsignal indicates the presence of a target transcript in said cell

The invention comprises a composition, comprising a non-replicativetransduction particle comprising a nucleic acid reporter construct, thenucleic acid reporter construct comprising a first promoter operativelylinked a reporter gene, wherein said first promoter is capable of beinginduced by an inducer protein endogenous in a bacterial cell.

The invention includes a method for detecting a presence of a bacterialcell in a sample comprising contacting said sample with anon-replicative transduction particle comprising nucleic acid reporterconstruct comprising a first promoter operatively linked to a reportergene, wherein said first promoter is capable of being induced by aninducer protein endogenous to said bacterial cell; and detecting thepresence or absence of an output signal from said reporter gene, whereinsaid presence of said output signal indicates the presence of saidbacterial cell in said sample.

In one embodiment, the first promoter is the same as an induciblepromoter operatively linked to a target nucleic acid molecule in saidbacterial cell.

The invention comprises a composition, comprising a non-replicativetransduction particle comprising a nucleic acid reporter construct, thenucleic acid reporter construct comprising a reporter gene that encodesa reporter molecule, the non-replicative transduction particle capableof entering a bacterial cell; and a caged substrate that exogenous tosaid bacterial cell that once un-caged is capable of reacting to saidreporter molecule in said cell.

The invention comprises a method for detecting a presence of a bacterialcell in a sample comprising contacting said sample with a cagedsubstrate and a non-replicative transduction particle comprising anucleic acid reporter construct, the nucleic acid reporter constructcomprising a reporter gene that encodes a reporter molecule, the cagedsubstrate exogenous to said cell that once un-caged is capable ofbinding to said reporter molecule in said bacterial cell; and detectingthe presence or absence of an output signal from said reporter molecule,wherein said presence of said output signal indicates the presence ofsaid bacterial cell in said sample.

In one embodiment, a target enzyme in said cell binds said cagedsubstrate to produce an un-caged substrate. In some embodiments, theun-caged substrate reacts with said reporter molecule to produce saidoutput signal.

The invention also includes a composition, comprising a non-replicativetransduction particle comprising a nucleic acid reporter construct, thenucleic acid reporter construct encoding a switchable molecule capableof binding to a target molecule in a bacterial cell to form a complex;and a substrate capable of penetrating said cell and binding saidcomplex to produce a detectable signal from said cell.

The invention includes a method for detecting a presence of a bacterialcell in a sample comprising contacting said sample with a substrate anda non-replicative transduction particle comprising a nucleic acidreporter construct encoding a switchable molecule, the switchablemolecule capable of binding a target molecule in said cell to form acomplex, the substrate capable of binding said complex to form asubstrate-bound complex; and detecting the presence or absence of anoutput signal from said substrate-bound complex, wherein said presenceof said output signal indicates the presence of said bacterial cell insaid sample. In one embodiment, the binding of said switchable moleculeto said target molecule produces a conformational change in saidswitchable molecule. In another embodiment, the conformational change insaid switchable molecule allows said substrate to bind to said complex.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawings, where:

FIG. 1 illustrates an example of the design and function of the silentmutation/complementation-based P1 plasmid packaging system, according toan embodiment of the invention.

FIG. 2 illustrates a schematic of the pGWP10001 vector, according to anembodiment of the invention.

FIG. 3 illustrates an example of the design and function of a pac-sitedeletion/complementation plasmid packaging system, according to anembodiment of the invention.

FIG. 4 illustrates a schematic of the pGW80A0001 vector, according to anembodiment of the invention.

FIG. 5 depicts the process for genomic island (GI) packaging by abacteriophage, according to an embodiment of the invention.

FIG. 6 depicts an example of the design and function of a GI-basedpackaging system, according to an embodiment of the invention.

FIG. 7 depicts the design and function of a GI-based packaging systemthat lacks the integrase gene, according to an embodiment of theinvention.

FIG. 8 depicts the design and function of a SaPIbov2-based packagingsystem that lacks the integrase gene, according to an embodiment of theinvention.

FIG. 9 depicts a system for the use of NRTPs for the detection ofinducers to target gene promoters within viable cells, according to anembodiment of the invention.

FIG. 10 depicts a reporter system that includes a reporter nucleic acidmolecule (e.g., plasmid) that is constructed for detecting VanR, theinducer of the promoter of the vancomycin resistance (vanA) gene inEnterococcus faecium (or E. faecalis), according to an embodiment of theinvention. The reporter plasmid carries a reporter gene that isoperatively linked to the vanA gene promoter.

FIG. 11 depicts a reporter system that includes a reporter nucleic acidmolecule constructed for detecting TcdD, the inducer of the promoters ofthe toxins A and B genes (tcdA and tcdB, respectively) of C. difficile,according to an embodiment of the invention. The reporter nucleic acidmolecule includes a reporter gene that is operatively linked to the tcdAgene promoter.

FIG. 12 depicts a reporter system that includes a reporter nucleic acidmolecule is constructed for detecting SarS, the inducer of the promoterof the Protein A gene (spa) in S. aureus, according to an embodiment ofthe invention. The reporter nucleic acid molecule includes the bacterialluciferase genes luxA and luxB operatively linked to the spa genepromoter (P_(spa)).

FIG. 13 shows a reporter system that comprises a system for thedetection of intracellular enzymes within viable cells that employscaged substrate molecules that can be un-caged by a target intracellularenzyme, according to an embodiment of the invention.

FIG. 14 depicts the design and function of a β-lactamase enzymedetection system, according to an embodiment of the invention.

FIG. 15 shows a reporter system for the detection of intracellularmolecules within viable cells that employs switchable molecules capableof generating a detectable signal upon their binding to a targetmolecule, according to an embodiment of the invention.

FIG. 16 depicts the design and function of abacteriophage/switchable-aptamer (SA)-based intracellular moleculereporter system, according to an embodiment of the invention.

FIG. 17 depicts an example of a system that uses a cis-repressionmechanism that can target the 5′ UTR (untranslated region) of a reportersequence on a reporter transcript, according to an embodiment of theinvention.

FIG. 18 shows an example of a system for detecting the presence of atarget transcript in a cell that is based on a cis-repression mechanismtargeting the ribosome binding site (RBS) of a reporter sequence in areporter transcript, according to an embodiment of the invention.

FIG. 19 illustrates an exemplary system for detecting the presence of atarget transcript in a cell that is based on a cis-repression mechanismtargeting the coding region (“AUG”) of a reporter sequence in a reportertranscript, according to an embodiment of the invention.

FIG. 20 illustrates an example system for detecting the presence of atarget transcript in a cell that is based on a repression mechanismusing an unstable reporter transcript, according to an embodiment of theinvention.

FIG. 21 shows the results of the transduction assay in which 36tetracycline-sensitive MRSA were exposed to transduction particlescarrying pGW80A0001 and then were spotted onto media plates containing 5ug/mL of tetracycline, according to an embodiment of the invention.

FIG. 22 illustrates the luminescence measured from 80 clinical isolatesof MRSA and 28 clinical isolates of methicillin sensitive S. aureus(MSSA) transduced with the transduction particle, according to anembodiment of the invention.

FIG. 23 shows the results of S. aureus growth at 4, 8, 16, 32, 64, and128 ug/mL of cefoxitin.

FIG. 24 shows the RLU values obtained by the NRTP assay in the presenceof 4, 8, 16, 32, 64, and 128 ug/mL cefoxitin. The x-axis in FIG. 24 isset at the MSSA RLU cutoff value.

FIG. 25 shows a secondary structure of the mecA transcript (SEQ IDNO:19) generated based on the lowest energy conformation calculated byMFold and visualized with VARNA.

FIG. 26 shows the terminal loop 23 (T23) of the mecA transcript (SEQ IDNO:23) that contains a YUNR consensus sequence.

FIG. 27 depicts a cis-repressing sequence (SEQ ID NO: 24) added to the5′ terminus of the luxAB genes and designed to form a stem-loopstructure that blocks the RBS sequence (“AAGGAA”) of the luxA gene.

FIG. 28 shows a diagram of base pairing between the target transcript(SEQ ID NO: 23) and the cis-repressing sequence of the reportertranscript (SEQ ID NO: 24).

FIG. 29 shows an example of a target mecA gene sequence (SEQ ID NO: 15),according to an embodiment of the invention.

FIG. 30 shows an exemplary mecA transcript sequence that can be used fordesigning a reporter transcript (SEQ ID NO:16), according to anembodiment of the invention.

FIG. 31 is an example of a luxAB gene loci DNA sequence that can be usedfor designing a reporter transcript (SEQ ID NO: 17), according to anembodiment of the invention.

FIG. 32 is an example of a luxAB transcript sequence that can be usedfor designing a reporter transcript (SEQ ID NO:18), according to anembodiment of the invention.

FIG. 33 is an example of a luxAB cis-repressed transcript sequence thatcan be used in a reporter transcript (SEQ ID NO:19), according to anembodiment of the invention.

FIG. 34 shows an example of a cell comprising a vector that encodes areporter transcript, where there is no endogenous mecA transcript in thecell, according to an embodiment of the invention.

FIG. 35 shows a vector introduced into a cell, where the vector encodesthe reporter transcript, which includes a cis-repressing sequence and areporter sequence (luxA and luxB genes). When the mecA transcriptpresent in the cell binds to the cis-repressing sequence, the inhibitoryhairpin loop opens up and the RBS for the luxA gene is exposed.Translation of the reporter sequences (luxA and luxB) can occur,resulting in the formation of a luxAB enzyme. The luxAB enzyme producesa detectable luminescent signal. In this manner, the transcript reportervector reports the presence of endogenous mecA transcripts within acell.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

As used herein, “reporter nucleic acid molecule” refers to a nucleotidesequence comprising a DNA or RNA molecule. The reporter nucleic acidmolecule can be naturally occurring or an artificial or syntheticmolecule. In some embodiments, the reporter nucleic acid molecule isexogenous to a host cell and can be introduced into a host cell as partof an exogenous nucleic acid molecule, such as a plasmid or vector. Incertain embodiments, the reporter nucleic acid molecule can becomplementary to a target gene in a cell. In other embodiments, thereporter nucleic acid molecule comprises a reporter gene encoding areporter molecule (e.g., reporter enzyme, protein). In some embodiments,the reporter nucleic acid molecule is referred to as a “reporterconstruct” or “nucleic acid reporter construct.”

A “reporter molecule” or “reporter” refers to a molecule (e.g., nucleicacid or protein) that confers onto an organism a detectable orselectable phenotype. The detectable phenotype can be colorimetric,fluorescent or luminescent, for example. Reporter molecules can beexpressed from reporter genes encoding enzymes mediating luminescencereactions (luxA, luxB, luxAB, luc, mc, nluc), genes encoding enzymesmediating colorimetric reactions (lacZ, HRP), genes encoding fluorescentproteins (GFP, eGFP, YFP, RFP, CFP, BFP, mCherry, near-infraredfluorescent proteins), nucleic acid molecules encoding affinity peptides(His-tag, 3X-FLAG), and genes encoding selectable markers (ampC, tet(M),CAT, erm). The reporter molecule can be used as a marker for successfuluptake of a nucleic acid molecule or exogenous sequence (plasmid) into acell. The reporter molecule can also be used to indicate the presence ofa target gene, target nucleic acid molecule, target intracellularmolecule, or a cell, as described herein. Alternatively, the reportermolecule can be a nucleic acid, such as an aptamer or ribozyme.

In some aspects of the invention, the reporter nucleic acid molecule isoperatively linked to a promoter. In other aspects of the invention, thepromoter can be chosen or designed to contribute to the reactivity andcross-reactivity of the reporter system based on the activity of thepromoter in specific cells (e.g., specific species) and not in others.In certain aspects, the reporter nucleic acid molecule comprises anorigin of replication. In other aspects, the choice of origin ofreplication can similarly contribute to reactivity and cross-reactivityof the reporter system, when replication of the reporter nucleic acidmolecule within the target cell contributes to or is required forreporter signal production based on the activity of the origin ofreplication in specific cells (e.g., specific species) and not inothers. In some embodiments, the reporter nucleic acid molecule forms areplicon capable of being packaged as concatameric DNA into a progenyvirus during virus replication.

As used herein, a “target transcript” refers to a portion of anucleotide sequence of a DNA sequence or an mRNA molecule that isnaturally formed by a target cell including that formed during thetranscription of a target gene and mRNA that is a product of RNAprocessing of a primary transcription product. The target transcript canalso be referred to as a cellular transcript or naturally occurringtranscript.

As used herein, the term “transcript” refers to a length of nucleotidesequence (DNA or RNA) transcribed from a DNA or RNA template sequence orgene. The transcript can be a cDNA sequence transcribed from an RNAtemplate or an mRNA sequence transcribed from a DNA template. Thetranscript can be protein coding or non-coding. The transcript can alsobe transcribed from an engineered nucleic acid construct.

A transcript derived from a reporter nucleic acid molecule can bereferred to as a “reporter transcript.” The reporter transcript caninclude a reporter sequence and a cis-repressing sequence. The reportertranscript can have sequences that form regions of complementarity, suchthat the transcript includes two regions that form a duplex (e.g., anintermolecular duplex region). One region can be referred to as a“cis-repressing sequence” and has complementarity to a portion or all ofa target transcript and/or a reporter sequence. A second region of thetranscript is called a “reporter sequence” and can have complementarityto the cis-repressing sequence. Complementarity can be fullcomplementarity or substantial complementarity. The presence and/orbinding of the cis-repressing sequence with the reporter sequence canform a conformation in the reporter transcript, which can block furtherexpression of the reporter molecule. The reporter transcript can formsecondary structures, such as a hairpin structure, such that regionswithin the reporter transcript that are complementary to each other canhybridize to each other.

“Introducing into a cell,” when referring to a nucleic acid molecule orexogenous sequence (e.g., plasmid, vector, construct), meansfacilitating uptake or absorption into the cell, as is understood bythose skilled in the art. Absorption or uptake of nucleic acidconstructs or transcripts can occur through unaided diffusive or activecellular processes, or by auxiliary agents or devices including via theuse of bacteriophage, virus, and transduction particles. The meaning ofthis term is not limited to cells in vitro; a nucleic acid molecule mayalso be “introduced into a cell,” wherein the cell is part of a livingorganism. In such instance, introduction into the cell will include thedelivery to the organism. For example, for in vivo delivery, nucleicacid molecules, constructs or vectors of the invention can be injectedinto a tissue site or administered systemically. In vitro introductioninto a cell includes methods known in the art, such as electroporationand lipofection. Further approaches are described herein or known in theart.

A “transduction particle” refers to a virus capable of delivering anon-viral nucleic acid molecule into a cell. The virus can be abacteriophage, adenovirus, etc.

A “non-replicative transduction particle” refers to a virus capable ofdelivering a non-viral nucleic acid molecule into a cell, but does notpackage its own replicated viral genome into the transduction particle.The virus can be a bacteriophage, adenovirus, etc.

A “plasmid” is a small DNA molecule that is physically separate from,and can replicate independently of, chromosomal DNA within a cell. Mostcommonly found as small circular, double-stranded DNA molecules inbacteria, plasmids are sometimes present in archaea and eukaryoticorganisms. Plasmids are considered replicons, capable of replicatingautonomously within a suitable host.

A “vector” is a nucleic acid molecule used as a vehicle to artificiallycarry foreign genetic material into another cell, where it can bereplicated and/or expressed.

A “virus” is a small infectious agent that replicates only inside theliving cells of other organisms. Virus particles (known as virions)include two or three parts: i) the genetic material made from either DNAor RNA molecules that carry genetic information; ii) a protein coat thatprotects these genes; and in some cases, iii) an envelope of lipids thatsurrounds the protein coat.

“MRSA” refers to Methicillin-resistant Staphylococcus aureus.

“MSSA” refers to Methicillin-sensitive Staphylococcus aureus.

The term “ameliorating” refers to any therapeutically beneficial resultin the treatment of a disease state, e.g., a disease state, includingprophylaxis, lessening in the severity or progression, remission, orcure thereof.

The term “in situ” refers to processes that occur in a living cellgrowing separate from a living organism, e.g., growing in tissueculture.

The term “in vivo” refers to processes that occur in a living organism.

The term “mammal” as used herein includes both humans and non-humans andinclude but is not limited to humans, non-human primates, canines,felines, murines, bovines, equines, and porcines.

“G,” “C,” “A” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, and uracil as a base, respectively.“T” and “dT” are used interchangeably herein and refer to adeoxyribonucleotide wherein the nucleobase is thymine, e.g.,deoxyribothymine. However, it will be understood that the term“ribonucleotide” or “nucleotide” or “deoxyribonucleotide” can also referto a modified nucleotide, as further detailed below, or a surrogatereplacement moiety. The skilled person is well aware that guanine,cytosine, adenine, and uracil may be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base may basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine may be replaced inthe nucleotide sequences of the invention by a nucleotide containing,for example, inosine. Sequences comprising such replacement moieties areembodiments of the invention.

As used herein, the term “complementary,” when used to describe a firstnucleotide sequence in relation to a second nucleotide sequence, refersto the ability of an oligonucleotide or polynucleotide comprising thefirst nucleotide sequence to hybridize and form a duplex structure undercertain conditions with an oligonucleotide or polynucleotide comprisingthe second nucleotide sequence, as will be understood by the skilledperson. Complementary sequences are also described as binding to eachother and characterized by binding affinities.

For example, a first nucleotide sequence can be described ascomplementary to a second nucleotide sequence when the two sequenceshybridize (e.g., anneal) under stringent hybridization conditions.Hybridization conditions include temperature, ionic strength, pH, andorganic solvent concentration for the annealing and/or washing steps.The term stringent hybridization conditions refers to conditions underwhich a first nucleotide sequence will hybridize preferentially to itstarget sequence, e.g., a second nucleotide sequence, and to a lesserextent to, or not at all to, other sequences. Stringent hybridizationconditions are sequence dependent, and are different under differentenvironmental parameters. Generally, stringent hybridization conditionsare selected to be about 5° C. lower than the thermal melting point(T_(m)) for the nucleotide sequence at a defined ionic strength and pH.The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the first nucleotide sequences hybridize to a perfectlymatched target sequence. An extensive guide to the hybridization ofnucleic acids is found in, e.g., Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes part I, chap. 2, “Overview of principles of hybridization and thestrategy of nucleic acid probe assays,” Elsevier, N.Y. (“Tijssen”).Other conditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotidecomprising the first nucleotide sequence to the oligonucleotide orpolynucleotide comprising the second nucleotide sequence over the entirelength of the first and second nucleotide sequence. Such sequences canbe referred to as “fully complementary” with respect to each otherherein. However, where a first sequence is referred to as “substantiallycomplementary” with respect to a second sequence herein, the twosequences can be fully complementary, or they may form one or more, butgenerally not more than 4, 3 or 2 mismatched base pairs uponhybridization, while retaining the ability to hybridize under theconditions most relevant to their ultimate application. However, wheretwo oligonucleotides are designed to form, upon hybridization, one ormore single stranded overhangs, such overhangs shall not be regarded asmismatches with regard to the determination of complementarity. Forexample, a dsRNA comprising one oligonucleotide 21 nucleotides in lengthand another oligonucleotide 23 nucleotides in length, wherein the longeroligonucleotide comprises a sequence of 21 nucleotides that is fullycomplementary to the shorter oligonucleotide, may yet be referred to as“fully complementary” for the purposes described herein.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs includes, but not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween two strands of a dsRNA, or between the antisense strand of adsRNA and a target sequence, between complementary strands of a singlestranded RNA sequence or a single stranded DNA sequence, as will beunderstood from the context of their use.

As used herein, a “duplex structure” comprises two anti-parallel andsubstantially complementary nucleic acid sequences. Complementarysequences in a nucleic acid construct, between two transcripts, betweentwo regions within a transcript, or between a transcript and a targetsequence can form a “duplex structure.” In general, the majority ofnucleotides of each strand are ribonucleotides, but as described indetail herein, each or both strands can also include at least onenon-ribonucleotide, e.g., a deoxyribonucleotide and/or a modifiednucleotide. The two strands forming the duplex structure may bedifferent portions of one larger RNA molecule, or they may be separateRNA molecules. Where the two strands are part of one larger molecule,and therefore are connected by an uninterrupted chain of nucleotidesbetween the 3′-end of one strand and the 5′-end of the respective otherstrand forming the duplex structure, the connecting RNA chain isreferred to as a “hairpin loop.” Where the two strands are connectedcovalently by means other than an uninterrupted chain of nucleotidesbetween the 3′-end of one strand and the 5′-end of the respective otherstrand forming the duplex structure, the connecting structure isreferred to as a “linker.” The RNA strands may have the same or adifferent number of nucleotides. The maximum number of base pairs is thenumber of nucleotides in the shortest strand of the duplex minus anyoverhangs that are present in the duplex. Generally, the duplexstructure is between 15 and 30 or between 25 and 30, or between 18 and25, or between 19 and 24, or between 19 and 21, or 19, 20, or 21 basepairs in length. In one embodiment the duplex is 19 base pairs inlength. In another embodiment the duplex is 21 base pairs in length.When two different siRNAs are used in combination, the duplex lengthscan be identical or can differ.

As used herein, the term “region of complementarity” refers to theregion on the antisense strand that is substantially complementary to asequence, for example a target sequence, as defined herein. Where theregion of complementarity is not fully complementary to the targetsequence, the mismatches are most tolerated in the terminal regions and,if present, are generally in a terminal region or regions, e.g., within6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.

The term percent “identity,” in the context of two or more nucleic acidor polypeptide sequences, refer to two or more sequences or subsequencesthat have a specified percentage of nucleotides or amino acid residuesthat are the same, when compared and aligned for maximum correspondence,as measured using one of the sequence comparison algorithms describedbelow (e.g., BLASTP and BLASTN or other algorithms available to personsof skill) or by visual inspection. Depending on the application, thepercent “identity” can exist over a region of the sequence beingcompared, e.g., over a functional domain, or, alternatively, exist overthe full length of the two sequences to be compared.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information.

The term “sufficient amount” means an amount sufficient to produce adesired effect, e.g., an amount sufficient to produce a detectablesignal from a cell.

The term “therapeutically effective amount” is an amount that iseffective to ameliorate a symptom of a disease. A therapeuticallyeffective amount can be a “prophylactically effective amount” asprophylaxis can be considered therapy.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise.

II. Lysogenic and Lytic Cycle of Viruses

Viruses undergo lysogenic and lytic cycles in a host cell. If thelysogenic cycle is adopted, the phage chromosome can be integrated intothe bacterial chromosome, or it can establish itself as a stable plasmidin the host, where it can remain dormant for long periods of time. Ifthe lysogen is induced, the phage genome is excised from the bacterialchromosome and initiates the lytic cycle, which culminates in lysis ofthe cell and the release of phage particles. The lytic cycle leads tothe production of new phage particles which are released by lysis of thehost.

Certain temperate phage can exhibit lytic activity, and the propensityfor this may vary with varying host bacteria. To illustrate thisphenomenon, the lytic activity of two temperate S. aureus phages on tenMRSA clinical isolates was examined via plaque assay (Table 1). Thephage φ11 exhibited lytic activity on 10 out of 10 clinical MRSAisolates and φ80α exhibited lytic activity on six of the 10 clinicalMRSA isolates. Thus, reporter assays relying on the natural lysogeniccycle of phages can be expected to exhibit lytic activity sporadically.

TABLE 1 Lytic activity (denoted by the letter “x”) of the S. aureustemperate phages ϕ11 and ϕ80α on ten clinical MRSA isolates MRSA isolateϕ11 ϕ80α 1 x 2 x 3 x x 4 x x 5 x x 6 x 7 x x 8 x 9 x x 10 x x

In addition, virus-based reporter assays, such as phage-based reporters,can suffer from limited reactivity (i.e., analytical inclusivity) due tolimits in the phage host range caused by host-based and prophage-derivedphage resistance mechanisms. These resistance mechanisms target nativephage nucleic acid that can result in the degradation or otherwiseinhibition of the phage DNA and functions. Such resistance mechanismsinclude restriction systems that cleave phage DNA and CRISPR systemsthat inhibit phage-derived transcripts.

Both lytic activity and phage resistance can be inhibitory to assaysbased on reporter phages. Lytic activity can inhibit signal bydestroying or otherwise inhibiting the cell in its ability to generate adetectable signal and thus affecting limits of detection by reducing theamount of detectable signal or preventing the generation of a detectablesignal. Phage resistance mechanisms can limit the host range of thephage and limit the inclusivity of the phage-based reporter, similarlyaffecting limits of detection by reducing the amount of detectablesignal or preventing the generation of a detectable signal. Both lyticactivity and phage resistance caused by the incorporation of phage DNAin a reporter phage can lead to false-negative results in assays thatincorporate these phage reporters.

III. Methods for Producing Non-Replicative Transduction Particles (NRTP)

A. Disruption/Complementation-Based Methods for ProducingNon-Replicative Transduction Particles

1) Silent Mutation/Complementation Packaging System

The invention includes methods for producing NRTPs using a silentmutation/complementation-based method.

This non-replicative transduction particle packaging system is based onintroducing a silent mutation into a component of the genome of a virusthat is recognized by the viral packaging machinery as the element fromwhich genomic packaging is initiated during viral production. Examplesof such an element include the pac-site sequence of pac-typebacteriophages and the cos-site sequence of cos-type bacteriophages.

Because these packaging initiation sites are often found within codingregions of genes that are essential to virus production, the silentmutation is introduced such that the pac-site is no longer recognized asa site of packaging initiation by the viral packaging machinery. At thesame time, the mutation does not disrupt the gene in which the site isencoded. By disrupting the packaging site sequence, the mutated virus isable to undergo a lytic cycle, but is unable to package its genomic DNAinto its packaging unit.

An exogenous reporter nucleic acid molecule, such as plasmid DNA, can beintroduced into a host cell that has been lysogenized with a viralgenome with a mutated packaging initiation site sequence. The exogenousreporter nucleic acid molecule can include a native packaging initiationsite sequence. The exogenous reporter nucleic acid molecule can beintroduced into the cell and replicated in the cell. When the mutatedvirus is undergoing a lytic cycle, the expressed viral packagingmachinery packages the exogenous reporter nucleic acid molecule with thenative packaging initiation site sequence into the viral packaging unit.The viral genome is not packaged into the packaging unit because itspackaging initiation site sequence has been mutated. In certainembodiments, the mutation in the packaging initiation site sequencecomprises a silent mutation that prevents cleavage of the packaginginitiation sequence, but does not disrupt the expression of the geneproduct that encompasses the packaging initiation site sequence. Thisproduces non-replicative transduction particles, e.g., viral structuralcomponents carrying the replicated exogenous nucleic acid molecule.

An example of such a system is based on the bacteriophage P1, a pac-typephage. In an embodiment, a plasmid including a native P1 pac site istransformed into a cell. The cell is lysogenized with a P1 prophagegenome. The P1 prophage genome includes a silent mutation in thepac-site sequence encoded within the pacA gene of P1. When the lyticcycle of the prophage is induced, the system results in the productionof P1-based transduction particles carrying the plasmid DNA. An exampleof a silent mutation that is suitable for this system is described inU.S. Pub. No. 2005/0118719, filed on Nov. 7, 2002, which is incorporatedby reference in its entirety. An example is also found in SEQ ID NO: 2,listed below (P1 pac-site with silent mutations, lower case letterssignify mutated bases).

FIG. 1 illustrates an example of the design and function of the silentmutation/complementation-based P1 plasmid packaging system 100,according to an embodiment of the invention. In this system, an E. colicell 101 is lysogenized with a P1 prophage 102 that includes a silentmutation in its packaging initiation site sequence (e.g., pac-site). Thecell is transformed with a plasmid containing the native pac-site 103,and the plasmid is replicated in the cell to form plasmid concatamers104. The plasmid can also include a reporter gene that encodes areporter molecule. When the lytic cycle of the P1 prophage is induced,the P1 prophage is excised from the bacterial genome and the P1structural components, such as capsid proteins, 105 are expressed. TheP1 structural components only package DNA that contains a nativepac-site (e.g., plasmid DNA), thus producing non-replicativetransduction particles carrying plasmid DNA 106 (e.g., a reporter gene).

An example vector for use in the silent mutation/complementation-basedP1 plasmid packaging system is shown in FIG. 2. Details about how toconstruct the strains and vectors of the silentmutation/complementation-based P1 plasmid packaging system are describedin detail in Example 1 below.

2) Deletion/Complementation-Based Packaging System

The invention includes methods for producing NRTPs using adeletion/complementation-based method.

This non-replicative transduction particle packaging system is based ondeletion of a component of the genome of a virus that is recognized bythe viral packaging machinery as the element from which genomicpackaging is initiated during viral production. Examples of such anelement include the pac-site sequence of pac-type bacteriophages and thecos-site sequence of cos-type bacteriophages. These packaging initiationsites are often found within coding regions of genes that are essentialto virus production. In some embodiments, the packaging initiation sitealone is deleted, which allows the mutated virus to undergo a lyticcycle but does not allow the virus to package its genomic DNA. Forexample, SEQ ID NO: 6 is an example of a P1 pacA gene with a deletedpac-site sequence (lower case letters indicate the deleted pac-sitesequence). In other embodiments, the entire gene comprising thepackaging initiation site is deleted. For example, SEQ ID NO: 8 showsthe deletion of the terS gene (lower case characters show the deletedsequence).

In one example, a cell's genome is lysogenized with a viral genome wherethe packaging initiation site has been deleted. A complementing plasmidis introduced into the cell, and the plasmid DNA includes a gene with apackaging initiation site sequence that complements the deletedpackaging initiation site sequence in the viral genome. When the mutatedvirus is undergoing a lytic cycle, the viral packaging proteins packagea replicon of the plasmid DNA into the packaging unit because of itspackaging initiation site, and non-replicative transduction particlesare produced carrying the replicated plasmid DNA.

In some embodiments, it is preferable that the deletion/complementationis designed such that there is no homology between the mutated virus DNAand the complementing exogenous DNA. This is because lack of homologybetween the mutated virus DNA and the complementing exogenous DNA avoidsthe possibility of homologous recombination between the two DNAmolecules that can result in re-introduction of a packaging sequenceinto the virus genome. To accomplish a lack of homology, one strategy isto delete the entire gene that contains the packaging initiation sitesequence from the virus genome and then complement this gene with anexogenous DNA molecule that contains no more than exactly the DNAsequence that was deleted from virus. In this strategy, thecomplementing DNA molecule is designed to express the gene that wasdeleted from the virus.

Another example of such a system is provided using the bacteriophageφ80α, a pac-type phage. The phage genome is lysogenized in a hostbacterial cell, and the phage genome includes a small terminase genewhere the pac-site of a pac-type prophage φ80α has been deleted. Aplasmid including a complementary small terminase gene with a nativepac-site is transformed into the cell. When the lytic cycle of thelysogenized prophage is induced, the bacteriophage packaging systempackages plasmid DNA into progeny bacteriophage structural components,rather than packaging the native bacteriophage DNA. The packaging systemthus produces non-replicative transduction particles carrying plasmidDNA.

FIG. 3 illustrates an example of the design and function of a pac-sitedeletion/complementation plasmid packaging system 300, according to anembodiment of the invention. A bacterial cell 301 is lysogenized with apac-type phage 302 that has its small terminase (terS) gene deleted. Thecell is transformed with a rolling circle replication plasmid 303 thatincludes a small terminase gene that complements the terS gene deletionin the phage. The small terminase gene contains the packaging initiationsite sequence, e.g., a pac-site. The plasmid 303 can also include areporter gene that encodes a reporter molecule.

A protein complex comprising the small terminase and large terminaseproteins is able to recognize and cleave a double-stranded DNA moleculeat or near the pac-site, and this allows the plasmid DNA molecule to bepackaged into a phage capsid. When the prophage in the cell is induced,the lytic cycle of the phage produces the phage's structural proteins304 and the phage's large terminase protein 305. The complementingplasmid is replicated, and the small terminase protein 306 is expressed.The replicated plasmid DNA 307 containing the terS gene (and thereporter gene) are packaged into phage capsids, resulting innon-replicative transduction particles carrying only plasmid DNA 308.FIG. 4 shows an example of a resulting vector used in the pac-sitedeletion/complementation plasmid packaging system. Further details aboutthe components and construction of pac-site deletion/complementationplasmid packaging system are in Example 2 below.

B. Pathogenicity Island-Based Packaging System

Pathogenicity islands (PTIs) are a subset of horizontally transferredgenetic elements known as genomic islands. There exists a particularfamily of highly mobile PTIs in Staphylococcus aureus that are inducedto excise and replicate by certain resident prophages. These PTIs arepackaged into small headed phage-like particles and are transferred atfrequencies commensurate with the plaque-forming titer of the phage.This process is referred to as the SaPI excision replication-packaging(ERP) cycle, and the high-frequency SaPI transfer is referred to asSaPI-specific transfer (SPST) to distinguish it from classicalgeneralized transduction (CGT). The SaPIs have a highly conservedgenetic organization that parallels that of bacteriophages and clearlydistinguishes them from all other horizontally acquired genomic islands.The SaPI1-encoded and SaPIbov2-encoded integrases are required for bothexcision and integration of the corresponding elements, and it isassumed that the same is true for the other SaPIs. Phage 80α can induceseveral different SaPIs, including SaPI1, SaPI2, and SaPIbov1, whereasφ11 can induce SaPIbov1 but neither of the other two SaPIs.

FIG. 5 depicts the natural process for genomic island (GI) packaging 500by a bacteriophage. In nature, a bacterial cell 501 lysogenized with asuitable prophage 503 and carrying a GI 504 can produce phage particlescarrying GI concatamers 512. In this process, when the phage is inducedinto its lytic cycle, the phage genome is excised (not shown) from thebacterial genome 502, which then expresses bacteriophage proteinsincluding capsid constituents 505 and the large terminase protein (TerL)506. Prophage induction also triggers GI excision via the expression ofthe GI integrase protein (int) 507. In a similar manner to the excisedphage genome (not shown), the GI circularizes 508, expresses its ownsmall terminase protein (TerS) 509, and begins to replicate forming a GIconcatamer 510. The phage TerL gene and GI TerS gene can then combinebind and cleave the GI concatamer via a pac-site sequence in the GIgenome, and the GI concatamer can then be packaged into phage capsids511 resulting in phage particles carrying GI concatamers 512.

In natural systems, as depicted in FIG. 5, the resulting lysate producedfrom phage production includes both native phage particles, as well asGI-containing phage particles. The native phage particles are a resultof packaging of the native phage genome due to recognition of thepac-site within phage genome concatamers.

1) Genomic Island (GI) Packaging System Design and Function

Methods of the invention for producing NRTPs include a GIbased-packaging system.

Compared to a plasmid packaging system, the natural GI-packaging systembenefits from the fact that the DNA that is packaged is derived from agenomic region within the bacterial genome and thus does not require themaintenance of a plasmid by the bacterial host.

In some embodiments, the invention includes a bacterial cell packagingsystem for packaging a reporter nucleic acid molecule into anon-replicative transduction particle, wherein the bacterial cellcomprises a lysogenized bacteriophage genome lacking a packaging gene,and a genomic island, cryptic phage, or other nucleic acid moleculerequiring a bacteriophage (e.g., a heper phage) for mobilization of thenucleic acid molecule and comprising a reporter nucleic acid moleculeand a packaging gene. Genomic island-based systems can be based on S.aureus Pathogenicity Islands (SaPIs), the E. coli criptic phage P4 andhelper phage P2, and the Enterococci criptic phage P7 and helper phageP1, for example.

GI-packaging systems can be exploited such that exogenous nucleic acidsequences are packaged by the bacteriophage. This can be accomplished byincorporating such exogenous nucleic acids sequences into the GI.

In order to eliminate the native phage from this process, the smallterminase gene of the prophage can be deleted. The small terminase genesequence contains the pac-site sequence of the native phage, and thisdeletion has the effect of preventing the packaging of native phage DNA.In other embodiments, only the pac site of the small terminase gene canbe deleted. The GI that will be packaged includes its own pac-site and asmall terminase gene that expresses a suitable small terminase protein,and only GI DNA will be amenable for packaging in this system.

FIG. 6 depicts an example of the design and function of a GI-basedpackaging system 600, according to an embodiment of the invention. Inthis system, a bacterial cell 601 has its genome lysogenized with asuitable prophage 603 that has its small terminase gene deleted, and thecell's genome 602 carries a GI 604. When the phage is induced into itslytic cycle, the phage genome is excised (not shown) from the bacterialgenome 602. The phage genome expresses bacteriophage proteins, includingcapsid constituents 605 and the large terminase protein (TerL) 606.Prophage induction also triggers GI excision via the expression of theGI integrase protein (int) 607. In a similar manner to the excised phagegenome (not shown), the GI circularizes 608 and expresses its own smallterminase protein (TerS) 609 and is replicated forming a GI concatamer610. The phage TerL gene and GI TerS gene can then combine, bind andcleave the GI concatamer via a pac-site sequence in the GI DNA. The GIconcatamer can then be packaged into phage capsids 611 resulting inphage particles carrying GI concatamers 612. In this system, phage DNAwill not be packaged into phage particles, since it lacks the terS genethat contains the phage's pac-site sequence, and thus cannot berecognized by the expressed GI TerS and phage TerL proteins.

When phage particles containing packaged GI DNA are administered to arecipient cell, the phage will bind to the recipient cell's surface andthen introduce the packaged GI DNA concatamer into the cell. Once insidethe cell, the GI can again express its integrase protein, and the GI canthen integrate into its specific site in the recipient cell's genome. Ifexogenous DNA sequences are included in the GI prior to packaging, thepackaging system thus allows for delivering exogenous DNA sequences to arecipient cell and integrating these exogenous DNA sequences into therecipient cell's genome.

2) GI-Based Packaging System Lacking Integrase

In another embodiment, the packaging system described above is designedsuch that packaged GI DNA cannot integrate into a recipient cell'sgenome. This can be accomplished by deleting the integrase gene in theGI and complementing the deletion by causing the expression of theintegrase gene in trans from the GI. In this manner, the integraseprotein is available for excision of the GI in the packaging host cell,and the GI DNA that has been packaged in a bacteriophage does notcontain the integrase gene and cannot express the integrase protein,thus preventing integration of the delivered GI.

FIG. 7 depicts the design and function of a GI-based packaging systemthat lacks the int gene 700, according to an embodiment of theinvention. In this system, a bacterial cell 701 is lysogenized with asuitable prophage that has had its small terminase gene deleted 703. Thecell's genome 702 carries a GI that has its integrase (int) gene deleted704 and also carries the deleted int gene operatively linked to asuitable promoter 705. The int gene can thus express the integraseprotein (Int) in trans from the GI 706. When the phage is induced intoits lytic cycle, the phage genome is excised (not shown) from thebacterial genome 702, which then expresses bacteriophage proteinsincluding capsid constituents 707 and the large terminase protein (TerL)708. Prophage induction also triggers GI excision via the expression ofthe integrase protein 707. In a similar manner to the excised phagegenome (not shown), the excised GI circularizes 709, expresses its ownsmall terminase protein (TerS) 710, and begins to replicate forming a GIconcatamer 711. The phage TerL gene and GI TerS gene can then combine,bind and cleave the GI concatamer via a pac-site sequence in the GI DNA,and the GI concatamer can then be packaged into phage capsids 712resulting in phage particles carrying GI concatamers 713. In thissystem, phage DNA will not be packaged since it lacks the terS gene thatcontains the phage's pac-site sequence and thus cannot be recognized bythe expressed GI TerS and phage TerL proteins.

When phage particles containing packaged GI DNA lacking the int gene areadministered to a recipient cell, the phage will bind to the recipientcell's surface and then introduce the packaged GI DNA concatamer intothe cell. Once inside the cell, the GI cannot express its integraseprotein due to the lack of the integrase gene and the GI cannot thenintegrate into its specific site in the recipient cell's genome. Ifexogenous DNA sequences are included in the GI prior to packaging, thepackaging system thus allows for delivering exogenous DNA sequences to arecipient cell and the delivered DNA sequences do not integrate into therecipient cell's genome at the specific site for GI integration.

3) Design and Function of SaPIbov2-Based Packaging Lacking Integrase

In some embodiments, the method of producing NRTPs employ a GI SaPIbov2and a bacteriophage φ11 in a GI-based packaging system. Alternativeembodiments can employ other SaPI GI's and other suitablebacteriophages, including the SaPI's SaPI1, SaPI2, SaPIbov1, andSaPIbov2 along with the bacteriophage 80α, and the SaPI's SaPIbov1 andSaPIbov2 along with the bacteriophage φ11. Based on the descriptionbelow, one of skill in the art would know how to develop a GI-basedpackaging system that does not lack the int gene, as described inSection II A.

FIG. 8 depicts the design and function of a SaPIbov2-based packagingsystem 800 that lacks the int gene, according to an embodiment of theinvention. In this system, a S. aureus cell 801 is lysogenized with φ11that has its small terminase gene deleted 803. The cell's genome 802carries SaPIbov2 that has its integrase (int) gene deleted 804 and alsocarries the deleted int gene operatively linked the constitutivelyexpressed PclpB gene promoter 805. The int gene can express theintegrase protein (Int) in trans from SaPIbov2 806. When the phage isinduced into its lytic cycle, the phage genome is excised (not shown)from the bacterial genome 802, which then expresses bacteriophageproteins including capsid constituents 807 and the large terminaseprotein (TerL) 808. Prophage induction also triggers SaPIbov2 excisionvia the expression of the integrase protein 806. In a similar manner tothe excised phage genome (not shown), the excised SaPIbov2 circularizes809, expresses its own small terminase protein (TerS) 810 and begins toreplicate forming a SaPIbov2 concatamer 811. The phage TerL gene andSaPIbov2 TerS gene can then combine bind and cleave the SaPIbov2concatamer via a pac-site sequence in the SaPIbov2 DNA and the SaPIbov2concatamer can then be packaged into phage capsids 812 resulting inphage particles carrying SaPIbov2 concatamers 813. In this system, phageDNA will not be packaged since it lacks the terS gene that contains thephage's pac-site sequence and thus cannot be recognized by the expressedSaPIbov2 TerS and phage TerL proteins.

IV. Reporters

In some embodiments, the NRTPs and constructs of the invention comprisea reporter nucleic acid molecule including a reporter gene. The reportergene can encode a reporter molecule, and the reporter molecule can be adetectable or selectable marker. In certain embodiments, the reportergene encodes a reporter molecule that produces a detectable signal whenexpressed in a cell.

In certain embodiments, the reporter molecule can be a fluorescentreporter molecule, such as, but not limited to, a green fluorescentprotein (GFP), enhanced GFP, yellow fluorescent protein (YFP), cyanfluorescent protein (CFP), blue fluorescent protein (BFP), redfluorescent protein (RFP) or mCherry, as well as near-infraredfluorescent proteins.

In other embodiments, the reporter molecule can be an enzyme mediatingluminescence reactions (luxA, luxB, luxAB, luc, ruc, nluc, etc.).Reporter molecules can include a bacterial luciferase, a eukaryoticluciferase, an enzyme suitable for colorimetric detection (lacZ, HRP), aprotein suitable for immunodetection, such as affinity peptides(His-tag, 3X-FLAG), a nucleic acid that function as an aptamer or thatexhibits enzymatic activity (ribozyme), or a selectable marker, such asan antibiotic resistance gene (ampC, tet(M), CAT, erm). Other reportermolecules known in the art can be used for producing signals to detecttarget nucleic acids or cells.

In other aspects, the reporter molecule comprises a nucleic acidmolecule. In some aspects, the reporter molecule is an aptamer withspecific binding activity or that exhibits enzymatic activity (e.g.,aptazyme, DNAzyme, ribozyme).

Reporters and reporter assays are described further in Section V herein.

V. NRTPs and Reporter Assays

A. Inducer Reporter Assay

The invention comprises methods for the use of NRTPs as reportermolecules for use with endogenous or native inducers that target genepromoters within viable cells. The NRTPs of the invention can beengineered using the methods described in Section III and below inExamples 1-6.

In some embodiments, the method comprises employing a NRTP as areporter, wherein the NRTP comprises a reporter gene that is operablylinked to an inducible promoter that controls the expression of a targetgene within a target cell. When the NRTP that includes the reporter geneis introduced into the target cell, expression of the reporter gene ispossible via induction of the target gene promoter in the reporternucleic acid molecule.

FIG. 9 depicts a genomic locus of a target cell 900 with two genes, agene encoding an inducer 902 and a target gene 903. Also depicted is areporter nucleic acid molecule 904 that includes a reporter gene 905that is operatively linked to the promoter 906 of the target gene of thetarget cell. The reporter nucleic acid molecule 904 can be introducedinto the cell via a NRTP. In the native cell, when the inducer gene 902is expressed and produces the inducer protein 907, the inducer protein907 is able to induce the target gene promoter 906 that is operativelylinked to the target gene, thus causing the expression of the targetgene and the production of the target gene product 908.

When the reporter nucleic acid molecule 904 is present within the targetorganism, the inducer 907 is also able to induce the target genepromoter 906 present within the reporter nucleic acid molecule 904, thuscausing the expression of the reporter gene 905 resulting in theproduction of a reporter molecule 909 capable of generating a detectablesignal.

Thus, the production of a detectable signal from the reporter molecule909 is indicative of the presence of the cell, based on the presence ofthe inducer protein 907 within a target cell.

1) VanR Reporter System

In one embodiment, the reporter system includes NRTP comprising areporter nucleic acid molecule (e.g., plasmid). The reporter nucleicacid molecule can be constructed for detecting VanR, the inducer of thepromoter of the vancomycin resistance (vanA) gene in Enterococcusfaecium (or E. faecalis). The reporter plasmid carries a reporter genethat is operatively linked to the vanA gene promoter.

FIG. 10 outlines the design and function of a VanR reporter system. FIG.10 depicts a region of the transposon Tn1546 1001 that may be present inE. faecium. The Tn1546 transposon can include the vanR inducer gene 1002and the vanA target gene 1003. Also depicted in the figure is a reporternucleic acid molecule 1004 that can be packaged in a NRTP and introducedinto the cell. The reporter nucleic acid molecule 1004 includes areporter gene 1005 that is operatively linked to a promoter P_(H) 1006that controls the expression of the vanHAX operon that includes the vanAgene. In the native cell, when the vanR gene 1002 is expressed andproduces the VanR protein 1007, VanR is able to induce P_(H) 1006 in theTn1546 transposon, thus causing the expression of the vanA gene and thusproducing the VanA protein 1008. When the reporter nucleic acid molecule1003 (vector) is present within the target organism, VanR is also ableto induce P_(H) 1006 within the reporter nucleic acid molecule 1003,thus causing the expression of a reporter molecule 1009. Thus, theproduction of a reporter molecule is indicative of the presence of VanRwithin a target cell.

Examples of promoters that are suitable for the development of a VREassay include: the vanA gene promoter and a vanB gene promoter. Arthur,M., et al., The VanS sensor negatively controls VanR-mediatedtranscriptional activation of glycopeptide resistance genes of Tn1546and related elements in the absence of induction. J. Bacteriol., 1997.179(1): p. 97-106.

2) TcdD reporter system

In another embodiment of this system, a reporter nucleic acid moleculeis introduced into a cell using a NRTP. The reporter nucleic acidmolecule can be constructed for detecting TcdD, the inducer of thepromoters of the toxins A and B genes (tcdA and tcdB, respectively) ofC. difficile. The reporter nucleic acid molecule includes a reportergene that is operatively linked to the tcdA gene promoter.

FIG. 11 outlines the design and function of a TcdD reporter system,according to an embodiment of the invention. FIG. 11 depicts a region ofthe transposon PaLoc 1101 that may be present in C. difficile. The PaLoctransposon may contain the tcdD gene 1102 and the tcdA target gene 1103.Also depicted in the figure is a reporter nucleic acid molecule 1104(e.g., vector) that is introduced into the cell using a NRTP. Thereporter nucleic acid molecule 1104 includes the reporter gene 1105 thatoperatively linked to the tcdA gene promoter (P_(tcdA)) 1106.

In the native cell, when the tcdD gene is expressed and produces theTcdD protein 1107, TcdD is able to induce P_(tcdA) 1106 in the PaLoctransposon 1101, thus causing the expression of the tcdA gene 1103 andthus producing the toxin A protein 1108.

When the reporter nucleic acid molecule 1104 is present within thetarget organism, TcdD is also able to induce P_(tcdA) 1106 within thereporter vector, thus causing the expression of a reporter molecule1109. Thus, the production of a reporter molecule 1109 is indicative ofthe presence of TcdD within a target cell.

Examples of promoters suitable for the development of a C. difficileassay include: the tcdA gene promoter and the tcdB gene promoter.Karlsson, S., et al., Expression of Clostridium difficile Toxins A and Band Their Sigma Factor TcdD Is Controlled by Temperature. Infect.Immun., 2003. 71(4): p. 1784-1793.

Target Cells and Inducers:

Target cells can include eukaryotic and prokaryotic cell targets andassociated inducers.

Vector Delivery Systems:

The delivery of the vector containing the recombinant DNA can byperformed by abiologic or biologic systems. Including but not limited toliposomes, virus-like particles, transduction particles derived fromphage or viruses, and conjugation.

3) Bacteriophage-Based SarS Reporter System

In another embodiment of the invention, a reporter nucleic acid moleculeis constructed for detecting SarS, the inducer of the promoter of theProtein A gene (spa) in S. aureus. The reporter nucleic acid moleculecan be introduced into the cell in a NRTP and includes the bacterialluciferase genes luxA and luxB operatively linked to the spa genepromoter (P_(spa)). The reporter nucleic acid molecule is delivered toS. aureus via a NRTP, for example. If SarS is present in the cell, itwill induce the expression of the luxAB genes, thus producing luciferaseenzyme that is capable of generating a luminescent signal.

FIG. 12 outlines the design and function of a SarS reporter system,according to one embodiment of the invention. FIG. 12 depicts a regionof the S. aureus genome 1201 that contain the sarS gene 1202 and spagene 1203. Also depicted in the figure is a reporter nucleic acidmolecule (e.g., vector) 1204 delivered by NRTP to the cell and thatincludes the luxAB reporter genes 1205 that operatively linked to thepromoter P_(spa) 1206 that controls the expression of the spa gene 1203.

In the native cell, when the sarS gene 1202 is expressed, producing SarSprotein 1207, the protein is able to induce P_(spa) 1206 in the S.aureus genome transposon, thus causing the expression of the spa gene1203 and producing the Protein A 1208.

When the reporter nucleic acid molecule 1204 is present within thetarget organism, SarS 1207 is also able to induce P_(spa) 1206 withinthe reporter nucleic acid molecule 1204, thus causing the expression ofluxAB resulting in the production of the luciferase enzyme 1209 that cangenerate a luminescent signal. Thus, the production of luciferase isindicative of the presence of SarS within a target cell.

B. Enzyme Reporter Assay

The invention comprises a system for the detection of intracellularenzymes within viable cells that employs caged substrate molecules thatcan be un-caged by a target intracellular enzyme, according to anembodiment of the invention.

FIG. 13 depicts the design and function of an intracellular enzymedetection system. A reporter molecule-expressing vector 1301 isdelivered to a target cell 1302 with a NRTP (not shown). The reportermolecule-expressing vector 1301 is able to penetrate the target cell1302 via the NRTP and deliver a reporter molecule gene 1303 into thetarget cell 1302, and a reporter molecule 1304 can then be expressedfrom the reporter molecule gene 1303. A caged substrate 1305 is alsoadded to the target cell 1302 and is able to penetrate into the targetcell 1302. If a target intracellular enzyme 1307 is present in thetarget cell 1306, the enzyme 1307 is able to remove the caging componentof the caged substrate 1305, thus producing an un-caged substrate 1308.The un-caged substrate 1308 can then react with the reporter molecule1304 inside of the cell 1302, and the product of this reaction resultsin a detectable signal 1309.

Target Cells and Enzymes:

Target cells can include eukaryotic and prokaryotic cell targets andassociated enzymes, including, for example, β-lactamases in S. aureus.

Vector Delivery Systems:

The delivery of the vector containing the recombinant DNA can byperformed by abiologic or biologic systems. Including but not limited toliposomes, virus-like particles, transduction particles derived fromphage or viruses, and conjugation.

Reporter Molecules and Caged Substrates:

Various reporter molecules and caged substrates can be employed as thosedescribed in Daniel Sobek, J. R., Enzyme detection system with cagedsubstrates, 2007, Zymera, Inc.

1) Bacteriophage-Based β-Lactamase Reporter

In one embodiment, a reporter molecule-expressing vector can be carriedby a NRTP, such that the vector can be delivered into a bacterial cell.The reporter molecule to be expressed can be Renilla luciferase, and thecaged substrate can be Renilla luciferin that is caged, such that aβ-lactamase enzyme that is endogenous to the target cell is able tocleave the caging compound from the caged luciferin and release un-cagedluciferin.

FIG. 14 depicts the design and function of a β-lactamase enzymedetection system, according to an embodiment of the invention. A Renillaluciferase-expressing vector carried by a bacteriophage-based NRTP 1401is added to a target S. aureus cell 1402. The Renillaluciferase-expressing vector is able to penetrate the target cell 1402using a NRTP comprising the vector. The NRTP delivers the Renillaluciferase gene 1403 into the target cell 1402, and Renilla luciferase1404 can then be expressed from its gene. Caged Renilla luciferin 1405is also added to the target cell 1402 and is able to penetrate into thetarget cell 1402. If an intracellular α-lactamase 1407 is present in thetarget cell 1402, the enzyme is able to remove the caging component ofthe caged luciferin 1406, thus producing an un-caged luciferin 1408. Theun-caged luciferin 1408 can then react with the Renilla luciferase 1404inside of the cell 1402, and the product of this reaction results inluminescence 1409.

In this manner, when a target cell that contains the β-lactamase isexposed to the NRTP and caged luciferin, the cell will exhibit aluminescent signal that is indicative of the presence of the β-lactamasepresent in the cell.

C. Intracellular Molecule Reporter

The invention includes a system for the detection of intracellularmolecules within viable cells that employs switchable molecules capableof generating a detectable signal upon their binding to a targetmolecule.

FIG. 15 depicts the design and function of a switchable molecule(SM)-based intracellular molecule detection system. A SM-expressingvector 1501 is delivered to a target cell 1502 in a NRTP. TheSM-expressing vector 1501 is able to penetrate the target cell 1502 anddeliver a SM gene 1503 into the target cell 1502. A SM protein 1504 canthen be expressed from the SM gene 1503. The SM protein 1504 can thenbind to a target molecule 1505 inside of the cell and thus forms anSM-target molecule complex 1506. The binding of the SM 1504 to thetarget molecule 1505 results in a conformational change in the SM 1504that makes the bound SM amenable to binding of a substrate. A substrate1508 is added to the cell 1507 and is able to penetrate into the cell1502. Bound SM inside of the cell 1502 is able to also bind thesubstrate, thus forming a SM-target molecule-substrate complex 1509.Finally, the binding of the substrate 1508 by the target molecule-boundSM has the effect of producing a detectable signal 1510. Thus adetectable signal generated by the system is indicative of the presenceof a target molecule inside of a cell.

Target Cells and Molecules:

Various eukaryotic and prokaryotic cell targets can be employed andswitchable aptamer-based SM's can be designed to target various nucleicacid and amino acid-based intracellular molecular targets as describedin Samie Jaffrey, J. P., Coupled recognition/detection system for invivo and in vitro use, 2010, Cornell University.

Vector Delivery Systems:

The delivery of the vector containing the recombinant DNA can byperformed by abiologic or biologic systems. Including but not limited toliposomes, virus-like particles, transduction particles derived fromphage or viruses, and conjugation.

1) Non-Replicative Transduction Particle/Switchable Aptamer-BasedIntracellular Molecule Reporter System

In one example of this method, a switchable molecule-expressing vectorcan be carried by a bacteriophage-based transduction particle such thatthe vector can be delivered into a bacterial cell. The switchablemolecule to be expressed can be a switchable aptamer that is designed toundergo a conformational change upon its binding to an intracellulartarget molecule. The conformational change allows the aptamer to thenbind a fluorophore that exhibits enhanced fluorescence when bound by theaptamer.

FIG. 16 depicts the design and function of abacteriophage/switchable-aptamer (SA)-based intracellular moleculereporter system. A SA-expressing vector carried by a NRTP 1601 is addedto a target cell 1602. The NRTP 1601 is able to deliver theSA-expressing vector and the SA-expressing gene 1603 into the targetcell 1602. An SA protein 1604 can then be expressed from the SA gene1603. The SA protein 1604 can then bind to a target molecule 1605 insideof the cell and thus form an SA-target molecule complex 1606. Thebinding of the SA 1604 to the target molecule 1605 results in aconformational change in the SA that makes the bound SA amenable tobinding of a fluorophore 1608. A fluorophore 1607 is added to the celland is able to penetrate into the cell 1608. Bound SA inside of the cellis able to also bind the fluorophore thus forming an SA-targetmolecule-fluorophore complex 1609. Finally, the binding of thefluorophore by the target molecule-bound SA has the effect of enhancingthe fluorescence of the fluorophore 1610. Thus, a detectable fluorescentsignal generated by the system is indicative of the presence of a targetmolecule inside of a cell.

D. Transcript Reporter Assay

The invention comprises a reporter assay comprising an antisenseRNA-based method for detecting target transcripts within viable cells bycausing the expression of a reporter molecule if a target transcript ispresent within a cell.

Certain intracellular methods in the art for inhibiting gene expressionemploy small interfering RNA, such as double-stranded RNA (dsRNA), totarget transcribed genes in cells. The dsRNA comprise antisense andsense strands that are delivered into or expressed in cells, and thestrands of the dsRNA act via a trans-acting inhibition mechanism, whereone strand (typically the antisense strand) binds to a target genesequence (RNA transcript) and prevents expression of the target genesequence. Double-stranded RNA molecules have been shown to block (knockdown) gene expression in a highly conserved regulatory mechanism knownas RNA interference (RNAi). WO 99/32619 (Fire et al.) discloses the useof a dsRNA of at least 25 nucleotides in length to inhibit theexpression of genes in C. elegans. dsRNA has also been shown to degradetarget RNA in other organisms, including plants (see, e.g., WO 99/53050,Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see,e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals(see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.).However, binding of a strand of the dsRNA to the target gene can benon-specific. If a similar mechanism were to be applied to a detectionsystem, this non-specific binding can result in high false positiverates, which make it unsuitable for the development of clinically usefuldetection systems.

Previous trans-acting inhibition mechanisms have been shown to beunsuitable for development of clinically useful detection systems. Forexample, some methods result in high levels of non-specific signals andup to 90% false positive rate, when achieving a 90% sensitivity of theassay. See U.S. Pat. No. 8,329,889. Certain methods forpost-transcriptional regulation of gene expression have been developedthat use a cis-repressed marker transcript, such as a green fluorescentprotein marker, where the ribosomal binding site of the marker isblocked by the cis-repressing sequence, along with a trans-activatingRNA transcript. When the trans-activating RNA transcript binds to thecis-repressed marker transcript, the hairpin structure of thecis-repressed marker transcript is altered, and the upstream ribosomebinding site of the marker gene is exposed, allowing transcription andexpression of the marker gene. However, these methods have notpreviously been used for the detection of endogenous transcripts, norsuccessful beyond a basic switching mechanism for controlling expressionof genes in cells.

1) Nucleic Acid Molecule Interactions and Mechanisms

The methods of the invention take advantage of the transcript-levelregulation mechanisms, including antisense RNA (asRNA) mechanism incells, to deliver nucleic acid molecules into cells. The antisensemechanism includes all forms of sequence-specific mRNA recognitionleading to reduced, eliminated, increased, activated, or otherwisealtered expression of a target transcript. See Good, L., TranslationRepression By Antisense Sequences. Cellular and Molecular Life Sciences,2003. 60(5): p. 854-861, and Lioliou, E., RNA-mediated regulation inbacteria: from natural to artificial systems, New Biotechnology. 2010.27(3): p. 222-235. Naturally occurring asRNAs are found in all threekingdoms of life, and they affect messenger RNA (mRNA) destruction,repression and activation, as well as RNA processing and transcription.See Sabine, B., Antisense-RNA regulation and RNA Interference.Biochimica et Biophysica Acta (BBA)—Gene Structure and Expression, 2001.1575(1-3): p. 15-25. This mechanism has been exploited in inhibitingprotein synthesis for therapeutic applications.

Antisense RNA is a single-stranded RNA that is complementary to amessenger RNA (mRNA) strand transcribed within a cell. asRNA may beintroduced into a cell to inhibit translation of a complementary mRNA bybase pairing to it and physically obstructing the translation machinery.Antisense RNA anneal to a complementary mRNA target sequence, andtranslation of the mRNA target sequence is disrupted as a result ofsteric hindrance of either ribosome access or ribosomal read through.

The antisense RNA mechanism is different from RNA interference (RNAi), arelated process in which double-stranded RNA fragments (dsRNA, alsocalled small interfering RNAs (siRNAs)) trigger catalytically mediatedgene silencing, most typically by targeting the RNA-induced silencingcomplex (RISC) to bind to and degrade the mRNA. Annealing of a strand ofthe dsRNA molecule to mRNA or DNA can result in fast degradation ofduplex RNA, hybrid RNA/DNA duplex, or duplex RNA resembling precursortRNA by ribonucleases in the cell, or by cleavage of the target RNA bythe antisense compound itself.

The RNAi pathway is found in many eukaryotes and is initiated by theenzyme Dicer, which cleaves long double-stranded RNA (dsRNA) moleculesinto short double stranded fragments of ˜20 nucleotides that are calledsiRNAs. Each siRNA is unwound into two single-stranded RNAs (ssRNA),namely the passenger strand and the guide strand. The passenger strandis degraded, and the guide strand is incorporated into the RNA-inducedsilencing complex (RISC). In post-transcriptional gene silencing, theguide strand base pairs with a complementary sequence in a messenger RNAmolecule, and cleavage is induced by a protein called Argonaute, thecatalytic component of the RISC complex.

In regards to the nucleic acid interactions of the mechanisms of theinvention, interactions between a reporter transcript and a targettranscript can rely on base pairings between loops present in bothtranscripts (e.g., “kissing complexes”), or between a loop and asingle-stranded (ss) region. In some cases, the kissing complexformation suffices for mediating the desired effect of the interaction,and in other cases, propagation of the primary contacts will lead to aninteraction resulting in the desired effect.

2) Mechanisms for Cis-Repression and Trans-Activation of Translation ofa Reporter Construct Via Transcript-Level Regulation

The following description illustrates transcript reporter systems basedon various repression/activation mechanisms that can be used, accordingto embodiments of this invention. In each of FIGS. 17-20, a vectorincludes a reporter construct comprising a reporter sequence, and theregions on the reporter construct are shown in each of the figures,including regions that can be targeted for repression by acis-repressing sequence. The description below provides non-limitingexamples of various inhibition mechanisms, including transcriptionattenuation, translation attenuation, and destabilization of thetranscript, and various activation mechanisms including conformationalchanges and cleavage.

FIG. 17 depicts an example of a system 1700 that uses a cis-repressionmechanism that can target the 5′ UTR (untranslated region) 1701 of thereporter sequence 1702 on a reporter transcript 1703. The regions withinthe reporter sequence 1702 (5′UTR (1701), RBS, Coding Region and 3′UTR)are also shown. The cis-repressing sequence 1705 is upstream of thereporter sequence and up to the 5′ UTR 1701 of the reporter sequence. AnRNA polymerase 1704 transcribes the sequence of the reporter construct1703 from the vector 1706.

At some point during transcription, the transcription process is stoppedby the formation of a transcription termination (TT) stem-loop structure1707 in the reporter transcript 1703, due to an interaction within thetranscribed cis-repressing sequence 1705. The transcription termination1707 structure stops 1708 the RNA polymerase 1704 from transcribing thevector 1706. In some embodiments, a transcription termination protein(e.g., NusA in E. coli) binds to RNA polymerase and/or to thetranscription termination 1707 structure to cease transcription of thereporter construct.

When a target transcript 1709 is present in the cell, the targettranscript 1709 binds to the reporter transcript 1703. In someembodiments, the binding between the target transcript and the reportertranscript is by base pairing of the nucleotides in each sequence. Theinteraction between the target transcript 1709 and the reportertranscript 103 causes the transcription termination (TT) stem-loopstructure 1707 to be cleaved 1710. Cleavage of the reporter transcript1703 can occur by a cellular enzyme, such as RNase III, for example. Inthis case, the secondary structure of a target transcript is analyzedfor the presence of an RNAse III consensus sequence among the ssRNAregions of the secondary structure, for example 5′-nnWAWGNNNUUN-3′ (SEQID NO: 20) or 5′-NAGNNNNCWUWnn-3′ (SEQ ID NO: 21) where “N” and “n” areany nucleotide and “W” is A or U and “N” indicates a relatively strictrequirement for Watson-Crick base pairing, while “n” indicates a minimalrequirement for base pairing. When such a consensus sequence is found ona target transcript, the loop of the transcription termination structure1707 can be designed to be complementary to said RNAse III consensussequence such that when the ssRNA in each RNA molecule hybridize, theRNAse III cleavage site is formed allowing for cleavage of thetranscription termination structure 1707. In the mecA transcript, loopT23, starting at nucleotide 1,404, has the sequence CAGAUAACAUUUU (SEQID NO: 22) that is suitable for such an approach.

In some embodiments, a cleavage site is engineered in the reporterconstruct, such that the reporter transcript is cleaved aftertranscription. The cleavage, in the example provided, can occurimmediately adjacent to the location of the loop in the transcriptionterminator structure. Transcription is re-initiated 1711 by the RNApolymerase 104. Cleavage of the transcription termination (TT) stem-loopstructure 1707 allows the remainder of the reporter sequence 1702 to betranscribed and subsequently translated. This results in the productionof a detectable or selectable marker from the translated reportermolecule.

In prokaryotes, the transcription termination structure 1707 involves aRho-independent mechanism with a stem-loop structure that is 7-20 basepairs in length, rich in cytosine-guanine base pairs and is followed bya chain of uracil residues. NusA binds to the transcription terminationstem-loop structure 1707 causing RNA polymerase to stall duringtranscription of the poly-uracil sequence. Weak Adenine-Uracil bondslower the energy of destabilization for the RNA-DNA duplex, allowing itto unwind and dissociate from the RNA polymerase. In eukaryotes, thetranscription termination structure 1707 is recognized by proteinfactors and involves cleavage of the new transcript followed bypolyadenylation.

FIG. 18 shows an example of a system 1800 for detecting the presence ofa target transcript in a cell that is based on a cis-repressionmechanism targeting the ribosome binding site (RBS) 1801 of the reportersequence 1702 in the reporter transcript 1703. The RBS 1801 is asequence of mRNA that is bound by the ribosome 1802 when initiatingprotein translation. The cis-repressing sequence 1705 is designed tobind to the RBS 1801 (e.g., the cis-repressing sequence 1705 iscomplementary to the RBS sequence 1801). The RBS 1801 binds to thecis-repressing sequence 1705 and becomes sequestered (inaccessible by aribosome 1802), preventing the translation of the reporter transcript1703. When a target transcript 109 from the cell binds to the reportertranscript 1703, the target transcript 1709 has a higher bindingaffinity for the RBS sequence 1801, and a conformational change occursin the reporter transcript 1703 in a manner that releases the bindingbetween the cis-repressing 1705 sequence and the RBS sequence 1801. Thisallows the ribosome 1802 to bind to the RBS 1801, thereby allowing fortranslation of the reporter transcript 1703.

FIG. 19 illustrates an exemplary system 1900 for detecting the presenceof a target transcript in a cell that is based on a cis-repressionmechanism targeting the coding region (“AUG”) 1901 of the reportersequence 1702 in the reporter transcript 1703. The cis-repressingsequence 1705 is constructed such that it binds with (e.g.,complementary to) the coding region 1901 of the reporter sequence 1702.The “AUG” start codon is shown as part of the coding region 1901. Thebinding of the cis-repressing sequence 1705 and the coding region 1901results in a conformation that leads to cleavage 1902 of the reporterconstruct 1703. Cleavage of the reporter transcript 1703 preventstranslation.

When a target transcript 1709 is present in the cell, the targettranscript 1709 binds to the cis-repressing sequence 1705 in a mannerthat causes a conformational change in the reporter transcript 1703.This conformational change prevents or removes the interaction betweenthe cis-repressing sequence 1705 and the coding region 1901 of thereporter sequence 1702, thereby allowing for translation of the reportersequence 1702.

FIG. 20 illustrates an example system 2000 for detecting the presence ofa target transcript in a cell that is based on a repression mechanismusing an unstable reporter transcript 2001. The reporter transcript 2001is designed to be unstable such that it forms an unstable conformationthat prevents the translation of the reporter transcript 2001. Areporter transcript 2001 is defined to be unstable if it is prone torapid degradation due to a variety of factors, such as activity ofexosome complexes or a degradosome. A target transcript 1709 in the cellbinds to a portion of the unstable reporter transcript 2001. In thisexample, the portion responsible for destabilizing the transcript islocated in the 3′ UTR 2005 of the reporter sequence, and the 3′ UTR 2005acts like the cis-repressing sequence of the reporter construct 1703.The binding of the target transcript 1709 with the 3′ UTR 2005 of thereporter sequence results in a cleaving event 2003 that stabilizes thereporter transcript 2001 and allows for translation 2004 of the reportertranscript 2001. Cleavage occurs upon binding of the target transcript1709 and serves to remove the portion of sequence that is responsiblefor destabilizing the transcript. In this example, the target transcript1709 binds to the 3′ UTR 405 of the reporter sequence, but the system400 can also be designed such that binding and cleavage occurs in the 5′UTR, upstream of the 5′ UTR, or downstream of the 3′ UTR. Binding andcleavage can occur anywhere outside of regions necessary for translationof the reporter sequence 1702.

In some embodiments, the cis-repressing sequence itself comprises twosequences that can bind to each other (e.g., complementary to eachother), and the conformation of the reporter transcript that resultsfrom the binding of the two sequences of the cis-repressing sequenceprevents translation of the reporter sequence in the reportertranscript.

3) Naturally Occurring and Synthetic Systems for Repression/ActivationMechanisms

Several naturally occurring and synthetically produced transcript-levelmechanisms have been described that demonstrate the individualmechanisms (i.e., conformational change and cleavage) employed in eachof the examples illustrated in FIGS. 17-20.

Transcription termination has been observed in antisense RNA(asRNA)-mediated transcriptional attenuation. In one example, twoloop—loop interactions between RNAIII/repR mRNA are subsequentlyfollowed by the formation of a stable duplex. This complex stabilizes aRho-independent terminator structure to arrest elongation by RNApolymerase (RNAP).

The RBS sequestration mechanism has been described via the developmentof a synthetic riboswitch system. In this system, a sequencecomplementary to a RBS is placed upstream of the RBS, allowing thepresence of a linker sequence between the two regions. Aftertranscription of the mRNA, the two complementary regions hybridize.creating a hairpin that prevents docking of the ribosome. To activatetranslation, a synthetic trans-activating RNA carrying the RBS sequencebinds to the hybridized RNA, allowing the RBS to be exposed andavailable for translation.

The prevention of translation due to the cleaving of RNA has also beendescribed in a natural system where the asRNA MicC targets a sequenceinside the coding region of ompD mRNA. The interaction, which ispromoted by Hfq, causes the cleaving of the mRNA by RNase E.

Yet another natural mechanism demonstrates a cleaving event to activatetranslation rather than inhibiting it. The E. coli GadY asRNA targetsthe intergenic region between two genes of the gadXW operon. Followingthe formation of a stable helix between GadY and the 3′UTR of gadX, anRNase cleavage occurs in the transcript and stabilizes gadX transcriptallowing for its translation.

4) Mechanism of Conformational Change by Cis-Repression of the ReporterSequence and by Binding of a Target Transcript

The general mechanisms employed in the invention are intermolecularnucleic acid molecule interactions that may result in two subsequentmechanisms: (1) a conformational change in the secondary structure ofthe nucleic acid molecules, and (2) a cleaving event. Described hereinare methods for designing reporter transcripts that can undergo aconformational change between a cis-repressed conformation and ade-repressed conformation, such that the conformational change isinduced by binding of a target transcript to the reporter transcript.

As described above, a reporter transcript can comprise a reportersequence and be designed such that translation of the reporter genesequence is blocked by cis-repression of the ribosome binding site (RBS)of the reporter gene.

In some embodiments, the following tools can be used for designing thereporter transcripts of the invention.

1) RNA secondary structure is calculated using secondary structureprogram, such as Mfold available at a server maintained by The RNAInstitute College of Arts and Sciences, University at Albany, StateUniversity of New York (Mfold web server for nucleic acid folding andhybridization prediction. Nucleic Acids Res. 31 (13), 3406-15, (2003)).

2) Intermolecular RNA interactions are calculated using a softwareprogram such as RNA-RNA InterACTion prediction using Integer Programming(RactIP) available at a server maintained by the Graduate School ofInformation Science, Nara Institute of Science and Technology (NAIST),Department of Biosciences and Informatics, Keio University Japan.

3) RNA secondary structure is visualized using Visualization Applet forRNA (VARNA), which is a Java lightweight Applet dedicated to drawing thesecondary structure of RNA.

A secondary structure of the target transcript can be generated based onthe lowest energy conformation calculated by MFold and visualized withVARNA.

ssRNA regions or target regions can be identified within the targettranscript that can be ideal for binding to a reporter transcript. Insome instances, the secondary structure of the target transcriptincludes a consensus sequence or loop sequence that can bind to aportion of the reporter sequence. For example, in the mecA transcript ofmethicillin-resistant S. aureus, there is a terminal loop that includesa consensus YUNR sequence (“UUGG”) that can be used to bind to acis-repressing sequence of a reporter transcript. Analysis of thesecondary structure of the target transcript can reveal these one ormore ssRNA regions that can be suitable for binding to a cis-repressingsequence. The cis-repressing sequence of the reporter transcript canthen be designed to bind to these one or more ssRNA regions.

In some embodiments, the cis-repressing sequence can be designed to bindto the RBS of the reporter sequence in the reporter transcript and forma stem-loop structure within the reporter transcript, such that thecis-repressing sequence blocks binding of an RNA polymerase to the RBSof the reporter sequence. Upon binding of the cis-repressing sequence tothe ssRNA region of the target transcript, the RBS of the reportersequence can be exposed and translation of the reporter sequence can beinitiated.

In some embodiments, the cis-repressing sequence of the reportertranscript can be designed to be positioned at the 5′ terminus of thereporter sequence and designed to generate a stem-loop structure in thereporter sequence, such that the RBS sequence of the reporter sequenceis blocked. The cis-repressing stem-loop structure can be designed toblock the RBS sequence based on the lowest energy conformation of thereporter transcript, as calculated by MFold and visualized with VARNA.The predicted inter-molecular interactions between the target transcriptand the cis-repressing sequence of the reporter transcript can becalculated by RactlP and visualized by VARNA. A diagram can be drawn tovisualize the base pairing between the target transcript and thecis-repressing sequence of the reporter transcript, as shown in FIG. 28below.

The interaction can include base pairing between 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, or 50 or more nucleotides in the target sequence andcis-repressing sequence. The complementary binding between the twosequences can be fully complementary, substantially complementary orpartially complementary. The base pairing can be across contiguousnucleotide sequences or regions within the target and cis-repressingsequences, for example, as shown in FIG. 28.

5) Cleavage Mechanisms for Cis-Repressed Transcripts or ReporterTranscripts

The general mechanisms employed in the invention are intermolecularnucleic acid molecule interactions that may result in two subsequentmechanisms: (1) a conformational change in the secondary structure ofthe nucleic acid molecules, and (2) a cleaving event. Described hereinare methods and systems for designing reporter transcripts that employ acleaving event.

In some embodiments, a cleaving mechanism can be employed in the systemand methods of the invention for cis-repression or for trans-activation.For example, as described above in FIGS. 17, 19 and 20, a system can bedesigned to take advantage of a cleaving mechanism by exposing a nucleicacid sequence of the reporter transcript to a cleaving enzyme (RNase) orsequestering a single-stranded sequence that is recognized by a sequencespecific RNAase.

In one example, an ribonuclease E (RNAse E) site can be designed in thereporter transcript (“*” indicates the cleaving site):(G,A)N(C,A)N(G)(G,U,A)*(A,U)(C,U)N(C,A)(C,A) (SEQ ID NO: 25). SeeKaberdin et al., Probing the substrate specificity of E. coli RNase Eusing a novel oligonucleotide-based assay. Nucleic Acids Research, 2003,Vol. 31, No. 16 (doi: 10.1093/nar/gkg690).

In a cis-repression system, a cis-repressing sequence can beincorporated in the design of a reporter transcript, such that whentranscribed, the conformation of the reporter transcript exposes asingle stranded region containing a sequence RNAse E recognition motifat the desired site to be cleaved. In some embodiments, the cleavagesite can be involved in repression of the transcription of the reportertranscript, for example, if the cleavage site is within the codingregion of the reporter gene.

For a trans-derepression system, the cis-repressed transcript can beengineered to bind to a target transcript, such that the interactioncauses a conformational change in the reporter transcript thatsequesters the single-stranded region containing the RNAse E site.

The system can be designed such that the cis-repressing mechanism is dueto a specific secondary structure generated by a conformation of thecis-repressing sequence, such as the transcription termination structuredescribed above. In this example, a cleaving event serves to de-repressthe reporter sequence. This can be accomplished by designing thecis-repressing sequence to interact with (bind to) a naturally-occurringplasmid or other cellular transcript, such that the interaction resultsin the generation of a single-stranded region containing the RNAse Esite that can be cleaved and thus removes the cis-repressing sequencefrom the reporter transcript.

In some embodiments, when a cleavage event is employed for expression ofthe reporter, the RNAse E site is designed to be outside of the codingregion of a reporter sequence with enough sequence length in the 5′ and3′ UTR in order to allow for a viable reporter transcript. In this case,the RNAse E site is designed to be at least 0, 1, 2, 3, 4, 5, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or more base pairsupstream of the start codon in prokaryotic systems and at least 18, 20,30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or more base pairsupstream of the start codon in eukaryotic systems or at least 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or more base pairsdownstream of the stop codon. In other embodiments, when a cleavageevent is employed for repression of the reporter, the RNAse E site isdesigned to be within the coding region of the reporter sequence orotherwise placed in order to inhibit expression of the reporter.

6) Transcripts

As described above, a transcript is a length of nucleotide sequence (DNAor RNA) transcribed from a DNA or RNA template sequence or gene. Thetranscript can be a cDNA sequence transcribed from an RNA template or anmRNA sequence transcribed from a DNA template. The transcript can betranscribed from an engineered nucleic acid construct. The transcriptcan have regions of complementarity within itself, such that thetranscript includes two regions that can form an intra-molecular duplex.One region can be referred to as a “cis-repressing sequence” that bindsto and blocks translation of a reporter sequence. A second region of thetranscript is called a “reporter sequence” that encodes a reportermolecule, such as a detectable or selectable marker.

The transcripts of the invention can be a transcript sequence that canbe 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25 nucleotides in length. In other embodiments, thetranscript can be at least 25, 30, 40, 50, 60, 70, 80, 90, 100, 500,1000, 1500, 2000, 3000, 4000, 5000 or more nucleotides in length. Thecis-repressing sequence and the reporter sequence can be the same lengthor of different lengths.

In some embodiments, the cis-repressing sequence is separated from thereporter sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45,50, 55, 60, or more spacer nucleotides.

7) Vectors

In another aspect, the transcripts (including antisense and sensesequences) of the invention are expressed from transcription unitsinserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG.(1996), 12:5-10; Skillern, A., et al., International PCT Publication No.WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, andConrad, U.S. Pat. No. 6,054,299). These sequences can be introduced as alinear construct, a circular plasmid, or a viral vector, includingbacteriophage-based vectors, which can be incorporated and inherited asa transgene integrated into the host genome. The transcript can also beconstructed to permit it to be inherited as an extrachromosomal plasmid(Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The transcript sequences can be transcribed by a promoter located on theexpression plasmid. In one embodiment, the cis-repressing and reportersequences are expressed as an inverted repeat joined by a linkerpolynucleotide sequence such that the transcript has a stem and loopstructure.

Recombinant expression vectors can be used to express the transcripts ofthe invention. Recombinant expression vectors are generally DNA plasmidsor viral vectors. Viral vectors expressing the transcripts can beconstructed based on, but not limited to, adeno-associated virus (for areview, see Muzyczka, et al., Curr. Topics Micro. Immunol. (1992)158:97-129)); adenovirus (see, for example, Berkner, et al.,BioTechniques (1998) 6:616), Rosenfeld et al. (1991, Science252:431-434), and Rosenfeld et al. (1992), Cell 68:143-155)); oralphavirus as well as others known in the art. Retroviruses have beenused to introduce a variety of genes into many different cell types,including epithelial cells, in vitro and/or in vivo (see, e.g., Eglitis,et al., Science (1985) 230:1395-1398; Danos and Mulligan, Proc. Natl.Acad. Sci. USA (1998) 85:6460-6464; Wilson et al., 1988, Proc. Natl.Acad. Sci. USA 85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad.Sci. USA 87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; vanBeusechem. et al., 1992, Proc. Natl. Acad. Sci. USA 89:7640-19; Kay etal., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol.150:4104-4115; U.S. Pat. Nos. 4,868,116; 4,980,286; PCT Application WO89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; andPCT Application WO 92/07573). Recombinant retroviral vectors capable oftransducing and expressing genes inserted into the genome of a cell canbe produced by transfecting the recombinant retroviral genome intosuitable packaging cell lines such as PA317 and Psi-CRIP (Comette etal., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc. Natl.Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be used toinfect a wide variety of cells and tissues in susceptible hosts (e.g.,rat, hamster, dog, and chimpanzee) (Hsu et al., 1992, J. InfectiousDisease, 166:769), and also have the advantage of not requiringmitotically active cells for infection.

Any viral vector capable of accepting the coding sequences for thetranscript(s) to be expressed can be used, for example, vectors derivedfrom adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g.,lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus,and the like. The tropism of viral vectors can be modified bypseudotyping the vectors with envelope proteins or other surfaceantigens from other viruses, or by substituting different viral capsidproteins, as appropriate.

For example, lentiviral vectors featured in the invention can bepseudotyped with surface proteins from vesicular stomatitis virus (VSV),rabies, Ebola, Mokola, and the like. AAV vectors featured in theinvention can be made to target different cells by engineering thevectors to express different capsid protein serotypes. Techniques forconstructing AAV vectors which express different capsid proteinserotypes are within the skill in the art; see, e.g., Rabinowitz J E etal. (2002), J Virol 76:791-801, the entire disclosure of which is hereinincorporated by reference.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingthe transcripts into the vector, and methods of delivering the viralvector to the cells of interest are within the skill in the art. See,for example, Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A(1988), Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1:5-14; Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al.,Nat. Genet. 33: 401-406, the entire disclosures of which are hereinincorporated by reference.

Viral vectors can be derived from AV and AAV. A suitable AV vector forexpressing the transcripts featured in the invention, a method forconstructing the recombinant AV vector, and a method for delivering thevector into target cells, are described in Xia H et al. (2002), Nat.Biotech. 20: 1006-1010. Suitable AAV vectors for expressing thetranscripts featured in the invention, methods for constructing therecombinant AV vector, and methods for delivering the vectors intotarget cells are described in Samulski R et al. (1987), J. Virol. 61:3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski Ret al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. Nos. 5,252,479;5,139,941; International Patent Application No. WO 94/13788; andInternational Patent Application No. WO 93/24641, the entire disclosuresof which are herein incorporated by reference.

The promoter driving transcript expression in either a DNA plasmid orviral vector featured in the invention may be a eukaryotic RNApolymerase I (e.g., ribosomal RNA promoter), RNA polymerase II (e.g.,CMV early promoter or actin promoter or U1 snRNA promoter) or generallyRNA polymerase III promoter (e.g., U6 snRNA or 7SK RNA promoter) or aprokaryotic promoter, for example the T7 promoter, provided theexpression plasmid also encodes T7 RNA polymerase required fortranscription from a T7 promoter. The promoter can also direct transgeneexpression to the pancreas (see, e.g., the insulin regulatory sequencefor pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA83:2511-2515)).

In addition, expression of the transcript can be precisely regulated,for example, by using an inducible regulatory sequence and expressionsystems such as a regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of transgene expression in cells or inmammals include regulation by ecdysone, by estrogen, progesterone,tetracycline, chemical inducers of dimerization, andisopropyl-beta-D-1-thiogalactopyranoside (IPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the dsRNA transgene.

Generally, recombinant vectors capable of expressing transcriptmolecules are delivered as described below, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of transcript molecules. Such vectors can be repeatedlyadministered as necessary. Once expressed, the transcript binds totarget RNA and modulates its function or expression. Delivery oftranscript expressing vectors can be systemic, such as by intravenous orintramuscular administration, by administration to target cellsex-planted from the patient followed by reintroduction into the patient,or by any other means that allows for introduction into a desired targetcell.

Transcript expression DNA plasmids are typically transfected into targetcells as a complex with cationic lipid carriers (e.g., Oligofectamine)or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiplelipid transfections for dsRNA-mediated knockdowns targeting differentregions of a single PROC gene or multiple PROC genes over a period of aweek or more are also contemplated by the invention. Successfulintroduction of vectors into host cells can be monitored using variousknown methods. For example, transient transfection can be signaled witha reporter, such as a fluorescent marker, such as Green FluorescentProtein (GFP). Stable transfection of cells ex vivo can be ensured usingmarkers that provide the transfected cell with resistance to specificenvironmental factors (e.g., antibiotics and drugs), such as hygromycinB resistance.

The delivery of the vector containing the recombinant DNA can byperformed by abiologic or biologic systems. Including but not limited toliposomes, virus-like particles, transduction particles derived fromphage or viruses, and conjugation.

8) Reporters for Transcript Assay

In some embodiments, the nucleic acid construct comprises a reportersequence (e.g., a reporter gene sequence). The reporter gene encodes areporter molecule that produces a signal when expressed in a cell. Insome embodiments, the reporter molecule can be a detectable orselectable marker. In certain embodiments, the reporter molecule can bea fluorescent reporter molecule, such as a green fluorescent protein(GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP),blue fluorescent protein (BFP), or red fluorescent protein (RFP). Inother embodiments, the reporter molecule can be a chemiluminescentprotein.

Reporter molecules can be a bacterial luciferase, an eukaryoticluciferase, a fluorescent protein, an enzyme suitable for colorimetricdetection, a protein suitable for immunodetection, a peptide suitablefor immunodetection or a nucleic acid that function as an apatamer orthat exhibits enzymatic activity.

Selectable markers can also be used as a reporter. The selectable markercan be an antibiotic resistance gene, for example.

9) Cells and Target Genes for Transcript Reporter Assay

Examples of cells that can be used for detection include Gram-positiveand Gram-negative bacteria, such as S. aureus, E. coli, K pneumoniae,etc., fungi such as Streptomyces coelicolor, and other eukaryotic cells,including cells from humans, other mammals, insects, invertebrates, orplants.

Target transcripts can include any endogenous transcript, whether codingor non-coding. Target transcripts can be derived from eukaryotic andprokaryotic cells, including, for example, mecA transcript in S. aureuscells (indicative of MRSA), the tcdB transcript in C. difficile(indicative of toxigenic C. diff), and HPV E6/E7 transcripts in cervicalepithelial cells (indicative of cervical cancer). Genes associated withinfectious agents, such as viruses, can be targets as well, includingHIV, HPV, etc. Other examples of target genes include non-coding RNAsuch as transfer RNA (tRNA) and ribosomal RNA (rRNA), as well as RNAssuch as snoRNAs, microRNAs, siRNAs, snRNAs, exRNAs, and piRNAs andncRNAs.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W. H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed.(Plenum Press) Vols A and B(1992).

Example 1: Silent Mutation/Complementation Packaging System

The following is an example of the design and construction of a silentmutation/complementation-based packaging system for producingnon-replicative transduction particles.

The materials used for developing the packaging system are listed below:

Bacterial Strains:

N1706, an E. coli K-12 P1 c1-100 Tn9 lysogen

Vectors:

Y14439 (pBHR1 backbone)

The following GenBank accession numbers (N.B., the sequences referred toby accession number are those listed in the database as of the prioritydate of this application) or SEQ ID NOs. can be used for the vectorbackbone and cassette sequences:

-   -   X06758 (bacterial luciferase genes luxAB)    -   SEQ ID NO:1 (Native P1 pac-site)    -   SEQ ID NO:3 (P1 lytic replicon containing the C1        repressor-controlled P53 promoter, the promoter P53 antisense,        the repL genes, and an in-frame deletion of the kilA gene)    -   SEQ ID NO:4 (Pblast promoter driving luxAB expression)

Construction of N1706(Pac): pacA Mutated Strain:

An exemplary sequence of a pacA mutated sequence is shown in SEQ ID NO:2, shown in the informal sequence listing below. The mutation can beaccomplished by constructing the mutated sequence via gene synthesis andthen replacing the native sequence in N1706 with the mutated sequencevia an allelic exchange approach.

Construction of the GWP10001 Reporter Vector:

The GWP10001 vector contains the pBHR1 origin of replication exhibitingbroad Gram-negative activity, two selectable markers for kanamycin andchloramphenicol, the native bacteriophage P1 pac-site sequence, the luxAand luxB genes are from Vibrio harveyi operatively linked to theconstitutive blasticillin promoter (Pblast), and the P1 lytic repliconcontaining the C1 repressor-controlled P53 promoter, the promoter P53antisense, the repL genes, and an in-frame deletion of the kilA gene.

FIG. 2 shows the resulting vector (GWP10001, SEQ ID NO:11), which can beconstructed in a variety of manners that are known to one of skill inthe art including obtaining the cassettes via PCR from their nativesources or via gene synthesis and assembly of the vector via traditionalrestriction enzyme-based cloning or alternative techniques such asGibson assembly.

Silent/Complementation Packaging System:

The packaging system includes the pacA mutant strain N1706(pac)complemented with the vector pGWP10001. As known to one of skill in theart, the manner of constructing this system can be accomplished bytransformation N1706(pac) with vector pGWP10001. The vector pGWP10001can be maintained in cultures of the transformed N1706(pac) by growingthe transformant in the presence of 50 ug/mL of kanamycin.

Production of Transduction Particles Carrying Plasmid DNA:

Non-replicative transduction particles carrying vector pGWP10001 can beproduced from N1706(pac) transformants via thermal induction at 42° C.Incubation at 42° C. results in induction of the P1 lytic cycle in whichthe prophage excises from the N1706 genome, produces phage structuralelements, and packages pGWP10001 concatameric DNA formed by the lyticreplicon in progeny phage particles, as depicted in FIG. 1. Theresulting cell lysate is then collected and contains non-replicativetransduction particles, each consisting of bacteriophage P1 particlescarrying a linear concatamer of pGWP10001 DNA.

Example 2: Deletion/Complementation Packaging System

The following is an example of the design and construction of adeletion/complementation-based packaging system for producingnon-replicative transduction particles.

The materials used for developing the packaging system are listed below:

Bacterial Strains:

RN4220 is a restriction defective S. aureus strain that is anon-lysogenic derivative of NCTC 8325 and is an efficient recipient forE. coli DNA. It was first described in Kreiswirth, B. N. et al., Thetoxic shock syndrome exotoxin structural gene is not detectablytransmitted by a prophage. Nature, 1983. 305(5936): p. 709-712.

RN10616 is derived by lysogenizing RN4220 with bacteriophage φ80α.Ubeda, C. et al., Specificity of staphylococcal phage and SaPI DNApackaging as revealed by integrase and terminase mutations. MolecularMicrobiology, 2009. 72(1): p. 98-108.

ST24 is derived from deleting the small terminase gene terS from thelysogenized bacteriophage φ80α in RN10616. Ubeda, C. et al., Specificityof staphylococcal phage and SaPI DNA packaging as revealed by integraseand terminase mutations. Molecular Microbiology, 2009. 72(1): p. 98-108.

Vectors:

Examples of plasmids that can be used as source plasmids for cassettes,in some embodiments of the invention are described in Charpentier, E.,et al., Novel Cassette-Based Shuttle Vector System for Gram PositiveBacteria. Appl. Environ. Microbiol., 2004. 70(10): p. 6076-6085.

The following GenBank accession numbers can be used for cassettesequences:

-   -   SEQ ID NO:5 (S. aureus pT181 plasmid origin or replication copy        number variant pT181cop-623 repC)    -   M21136 (tetA(M))    -   SEQ ID NO:12 (P_(clpB) promoter sequence)    -   SEQ ID NO:9 ((φ11 small terminase (terS) gene sequence)    -   L09137 (amp ColE1 ori)    -   X06758 (luxAB)    -   M62650 (Transcription Termination)

terS Deletion:

The construction of the terS knockout strain ST24 can be accomplishedvia an allelic-exchange-based strategy resulting in an in-frame deletionremoving most of the coding sequence of the φ80α small terminase gene.The details of this strategy are described in Ubeda, C. et al.,Specificity of staphylococcal phage and SaPI DNA packaging as revealedby integrase and terminase mutations. Molecular Microbiology, 2009.72(1): p. 98-108.

An exemplary sequence of a terS knockout strain is shown in SEQ IDNO:13, (shown in the sequence listing below). SEQ ID NO:13 is a RN10616genomic sequence loci showing the φ80α terS deletion andcomplementation.

Vector Construction:

The GW80A0001 vector is an E. coli/S. aureus shuttle vector. The vectorcontains S. aureus (pT181cop-623 repC) and E. coli (ColE1ori) origins ofreplication, the selectable markers for ampicillin (amp) andtetracycline (tet(M)) resistance for selection in E. coli and S. aureus,respectively, the φ11 small terminase (terS) gene sequence that includesits own promoter, the luxA and luxB genes are from Vibrio harveyioperatively linked to the constitutive S. aureus P_(clpB) promoter, anda transcription termination sequence (TT).

FIG. 4 shows the resulting vector (pGW80A0001, SEQ ID NO:14), which canbe constructed in a variety of manners that are known to one of skill inthe art. In one example, the tet(M) cassette and luxAB genes can beobtained via PCR amplification from the publically available pCN36 andpCN58 vectors (Charpentier, E., et al.). P_(clpB) can be obtained fromPCR amplification from S. aureus RN4220 and terS can be obtained via PCRamplification from RN10616. A vector backbone can be obtained byremoving the ermC gene from the publically available vector pCN48(Charpentier, E., et al.), and the various components of the finalvector pGW80A0001 can be assembled onto this vector backbone viaappropriately designed restriction enzyme-based cloning.

Deletion/Complementation Packaging System:

The packaging system can include the terS knockout strain ST24complemented with the vector pGW80A0001 to generate strain GW24. Asknown to one of skill in the art, the manner of constructing this systemcan be accomplished by transformation ST24 with vector pGW80A0001. Thevector pGW80A0001 can be maintained in cultures of the transformed ST24by growing the transformant in the presence of 5 ug/mL of tetracycline.

Production of Transduction Particles Carrying Plasmid DNA:

Non-replicative transduction particles carrying vector pGW80A0001 can beproduced from GW24 via a Mitomycin C-induction method that was firstdemonstrated in E. coli and is now a standard technique for obtainingprophages from lysogenized bacteria. Otsuji, N. et al., Induction ofPhage Formation in the Lysogenic Escherichia coli K-12 by Mitomycin C.Nature, 1959. 184(4692): p. 1079-1080. This prophage induction methodresults in induction of the φ80α lytic cycle in which the prophageexcises from the GW24 genome, produces phage structural elements, andpackages pGW80A0001 concatameric DNA in progeny phage particles, asdepicted in FIG. 2. The resulting cell lysate is then collected andcontains non-replicative transduction particles, each consisting ofbacteriophage φ80α particles carrying a linear concatamer of pGW80A0001DNA.

Example 3: SaPIbov2-Based Packing System Lacking Integrase

The following is an example of the design and construction of aSaPIbov2-based packaging system for producing non-replicativetransduction particles.

The materials used for developing the packaging system are listed below:

The following materials can be used to develop a SaPIbov2-basedpackaging system lacking integrase.

Bacterial Strains:

RN451 is a S. aureus strain lysogenized with bacteriophage φ11.

JP2131 is RN451 that has been lysogenized with SaPIbov2. See Maiques, E.et al., Role of Staphylococcal Phage and SaPI Integrase in Intra- andInterspecies SaPI Transfer. J. Bacteriol., 2007. 189(15): p. 5608-5616.

JP2488 is strain JP2131 in which the int gene has been deleted fromSapIbov2 (SaPIbov2Δint). Maiques, E. et al., Role of StaphylococcalPhage and SaPI Integrase in Intra-and Interspecies SaPI Transfer. J.Bacteriol., 2007. 189(15): p. 5608-5616.

Bacteriophage:

Bacteriophage φ11 can be obtained from S. aureus strain RN0451 via aMitomycin C-induction method that was first described in E. coli and isnow a standard technique for obtaining prophages from lysogenizedbacteria. Otsuji, N. et al., Induction of Phage Formation in theLysogenic Escherichia coliK-12 by Mitomycin C. Nature, 1959. 184(4692):p. 1079-1080.

Promoters:

P_(clpB) can be used as a promoter in this example. The clpB genepromoter is a constitutive promoter used for controlling the expressionof the int gene. The S. aureus clpB (P_(clpB)) gene promoter sequencewas first described in 2004. Frees, D., et al., Clp ATPases are requiredfor stress tolerance, intracellular replication and biofilm formation inStaphylococcus aureus. Molecular Microbiology, 2004. 54(5): p.1445-1462. It was also first employed for controlling the geneexpression in a plasmid in 2004. Arnaud, M., A. Chastanet, and M.Debarbouille, New Vector for Efficient Allelic Replacement in NaturallyNontransformable, Low-GC-Content, Gram-Positive Bacteria. Appl. Environ.Microbiol., 2004. 70(11): p. 6887-6891. The promoter can be obtainedfrom S. aureus RN4220 using primers described in 2004. Id.

Production of φ11 SaPIbov2Δint Co-Lysogen (RN451(φ11 SaPIbov2Δint)):

The strain JP2488(φ11 SaPIbov2Δint) can be produced by lysogenizingJP2488 with φ11.

Deletion of φ11 terS (RN451(φ11ΔterS SaPIbov2Δint)):

The strain RN451(φ11ΔterS SaPIbov2Δint) can be produced by deleting theφ11 terS gene from RN451(φ11SaPIbov2Δint), as described in Tormo, M. A.et al., Staphylococcus aureus Pathogenicity Island DNA Is Packaged inParticles Composed of Phage Proteins. J. Bacteriol., 2008. 190(7): p.2434-2440.

Incorporation of P_(clpB)-int into S. aureus Genome (RN451(φ11ΔterSSaPIbov2Δint P_(clpB)-int)):

RN451(φ11ΔterS SaPIbov2Δint P_(clpB)-int) can be produced by firstfusing P_(clpB) and int via standard molecular biology techniques theninserting the P_(clpB)-int fusion into the genome of RN451(φ11ΔterSSaPIbov2Δint) and then selecting clones that have P_(clpB)-int insertedoutside of the φ11 and SaPIbov2 regions.

Production of φ11 Particles Carrying Only SaPIbov2Δint P_(clpB)-intConcatamers:

φ11 particles carrying only SaPIbov2Δint P_(clpB)-int concatamers can beproduced via mitomycin-C induction of RN451(∠11ΔterS SaPIbov2ΔintP_(clpB)-int), as described by Otsuji, N. et al., Induction of PhageFormation in the Lysogenic Escherichia coliK-12 by Mitomycin C. Nature,1959. 184(4692): p. 1079-1080. The cell lysate contains non-replicativetransduction particles, each consisting of bacteriophage φ11 structuralproteins carrying a linear concatamer of GI-derived DNA.

One of skill in the art will understand how to construct the NRTPs ofthe invention using the above-referenced materials and well-knownmolecular biology and genetic techniques in the art.

Example 4: terS Deletion/Complementation-Based SarS ReporterTransduction Particles

The following is an example of an inducer reporter-based SarS reportersystem that employs a terS deletion/complementation-basednon-replicative transduction particle.

Reporter Gene:

Bacterial luciferase (luxAB). The luxA and luxB genes are from Vibrioharveyi. They lack a transcriptional promoter and each contains theirown ribosomal binding site.

Spa gene promoter (P_(spa)): The spa gene promoter will be used forcontrolling the expression of the luxAB genes.

Construction of P_(spa)-luxAB Fusion:

The luxAB genes can be fused to the P_(spa) promoter sequence such thatthe luxAB genes are operatively linked to the P_(spa) promoter.

Construction of the luxAB-Expressing Reporter Vector

The luxAB-expressing reporter vector can be constructed via standardmolecular biological techniques by incorporating the P_(Spa)-luxABfusion product into the MCS of an E. coli/S aureus shuttle vectordescribed below.

E. coli/S. aureus shuttle vector that carries a S. aureus (pT181cop-623repC) and E. coli (ColE1ori) origins of replication, genes forampicillin (amp) and tetracycline (tet(M)) resistance, the φ11 smallterminase (terS) gene under the control of a constitutive promoter(P_(clpB)), a multiple cloning site (MCS), and a transcriptiontermination sequence (TT).

GenBank accession numbers for cassette sequences:

J01764 (pT181 replicons)

M21136 (tetA(M))

Accession number not yet available (P_(clpB))

AF424781 REGION: 16526.16966 (terS)

L09137 (amp ColE1 ori)

M62650 (TT)

Propagation of the vector for conducting in vitro manipulations and forverification of manipulations can be accomplished via the E. coli Top 10and the final modified vector can then be introduced into S. aureusRN0451ΔterS. Transduction particles carrying shuttle vector can beproduced from the RN0451ΔterS transformants via a Mitomycin C-inductionmethod that was first described in E. coli 1959 and is now a standardtechnique for obtaining prophages from lysogenized bacteria. Otsuji, N.,et al., Induction of Phage Formation in the Lysogenic EscherichiacoliK-12 by Mitomycin C. Nature, 1959. 184(4692): p. 1079-1080. The celllysate is then collected and contains non-replicative transductionparticles each consisting of bacteriophage φ11 structural proteinscarrying a linear concatamer of plasmid DNA capable of reporting on thepresence of SarS in target S. aureus cells.

Example 5: terS Deletion/Complementation-Based β-lactamase ReporterTransduction Particles

The following is an example of an intracellular enzyme reporter-basedβ-lactamase reporter system that employs a terSdeletion/complementation-based non-replicative transduction particle.

Reporter Gene:

Renilla luciferase (ruc)

Promoter:

The promoter can be P_(blaZ). The constitutive beta-lactamase promotercan be used for driving the expression of the ruc gene.

Caged Substrate:

Caged coelenterazine-phosphate as described in Daniel Sobek, J. R.,Enzyme detection system with caged substrates, 2007, Zymera, Inc.

Construction of P_(blaZ)-Ruc Fusion:

The ruc genes can be fused to the P_(blaZ) promoter sequence such thatthe ruc genes are operatively linked to the P_(blaZ) promoter.

Construction of the Ruc-Expressing Reporter Vector:

The ruc-expressing reporter vector can be constructed via standardmolecular biological techniques by incorporating the P_(blaz)-ruc fusionproduct into the MCS of the shuttle vector depicted in Section V, A, 3),i) above.

Propagation of the vector for conducting in vitro manipulations and forverification of manipulations can be accomplished via the E. coli Top 10and the final modified vector can then be introduced into S. aureusRN0451ΔterS. Transduction particles carrying shuttle vector can beproduced from the RN0451ΔterS transformants via a Mitomycin C-inductionmethod that was first described in E. coli 1959 and is now a standardtechnique for obtaining prophages from lysogenized bacteria. Otsuji, N.,et al., Induction of Phage Formation in the Lysogenic EscherichiacoliK-12 by Mitomycin C. Nature, 1959. 184(4692): p. 1079-1080. The celllysate is then collected and contains NRTPs each consisting ofbacteriophage φ11 structural proteins carrying a linear concatamer ofplasmid DNA capable of expressing Renilla luciferase within viable S.aureus cells within the φ11 host range.

Example 6: terS Deletion/Complementation-Based Intracellular MoleculeReporter Transduction Particles

The following is an example of an intracellular molecule reporter-basedreporter system that employs a terS deletion/complementation-basednon-replicative transduction particle.

Promoter:

The promoter can be P_(blaz). The constitutive beta-lactamase promotercan be used for driving the expression of the ruc gene.

Switchable Aptamer:

Switchable aptamers can be designed and constructed as described inSamie Jaffrey, J. P., Coupled recognition/detection system for in vivoand in vitro use, 2010, Cornell University.

Fluorophore Substrate:

Corresponding fluorophore substrates in conjunction with the aboveswitchable aptamers can be designed and constructed as described inSamie Jaffrey, J. P., Coupled recognition/detection system for in vivoand in vitro use, 2010, Cornell University.

Construction of P_(blaZ)-SA Fusion:

The SA gene can be fused to the P_(blaZ) promoter sequence such that theSA gene is operatively linked to the P_(blaZ) promoter.

Construction of the SA-Expressing Reporter Vector:

The SA-expressing reporter vector can be constructed via standardmolecular biological techniques by incorporating the P_(blaz)-SA fusionproduct into the MCS of the shuttle vector depicted in Example 4 above.Propagation of the vector for conducting in vitro manipulations and forverification of manipulations can be accomplished via the E. coli Top 10and the final modified vector can then be introduced into S. aureusRN0451ΔterS. Transduction particles carrying shuttle vector can beproduced from the RN0451ΔterS transformants via a Mitomycin C-inductionmethod that was first described in E. coli 1959 and is now a standardtechnique for obtaining prophages from lysogenized bacteria. Otsuji, N.et al., Induction of Phage Formation in the Lysogenic Escherichia coliK-12 by Mitomycin C. Nature, 1959. 184(4692): p. 1079-1080. The celllysate is then collected and contains non-replicative transductionparticles each consisting of bacteriophage φ11 structural proteinscarrying a linear concatamer of plasmid DNA capable of expressing the SAwithin viable S. aureus cells within the φ11 host range.

Example 7: Non-Replicative Transduction Particle-Based Reporter System

The non-replicative transduction particles described above can be usedin a reporter system for detecting the presence of viable bacteria viathe expression of a reporter molecule (e.g. luxAB). When thistransduction particle introduces a reporter vector (e.g. pGW80A0001)into a cell within the host range of the transduction particle, cells inwhich the promoter (e.g. P_(clpB)) is recognized by the cellstranscription machinery are able to drive the expression of the reportermolecule within that cell.

To test the functionality of non-replicative transduction particles asreporters for detecting the presence of S. aureus cells, variousMSSA/MRSA reporter assays were developed. In an embodiment, anon-replicative transduction particle was developed from a S.aureus-specific bacteriophage, and the bacterial luciferase genes luxABunder the control of a constitutive promoter were incorporated. When thenon-replicative transduction particle delivered the reporter nucleicacid into S. aureus, the constitutive promoter expressed luxAB suitablefor reporting on the presence of a viable S. aureus.

In addition, the antibiotic cefoxitin was added prior to, simultaneouslywith, or after the addition of the transduction particles to a samplecontaining S. aureus cells. If the cells were not phenotypicallyresistant to cefoxitin (i.e., were not MRSA), luminescence was decreasedor eliminated, indicating that the cells were MSSA. If, however, thecells were phenotypically resistant to cefoxitin (i.e., were MRSA),increased or detectable luminescence was observed, indicating that thecells were MRSA.

Non-Replicative Transduction Particle-Based Viable Cell Reporter AssayFunction

The function of the non-replicative transduction particle as a reporterwas assayed. The transduction host range of the bacteriophage φ80α-basednon-replicative transduction particle was examined in 101 clinical MRSAisolates. The transduction assay was conducted by exposing cultures ofeach bacterial isolate grown in modified TSB to GW24 cell lysatecontaining the non-replicative transduction particles and culturing themixture on solid media containing tetracycline.

In this example, the non-replicative transduction particle carried atetracycline selectable marker. Cells transduced with thenon-replicative transduction particles were expected to be resistant totetracycline. In addition, transduction was examined via luminescenceassay by exposing each bacterial isolate in liquid culture to celllysate containing the non-replicative transduction particles andevaluating the mixture for bacterial luciferase luminescence activityafter an incubation period.

The transduction assay showed that the φ80α-based non-replicativetransduction particle was able to transduce all of the 101 clinicalisolates of MRSA and none of the non-S. aureus Staphylococci.

FIG. 21 shows the results of the transduction assay in which 36tetracycline-sensitive MRSA were exposed to transduction particlescarrying pGW80A0001 and then were spotted onto media plates containing 5ug/mL of tetracycline. The results show that all 36 MRSA strains grew onthe media containing tetracycline due to transduction with pGW80A0001.Control experiments in which MRSA isolates were spotted ontotetracycline containing media without exposure to transduction particlesshowed no growth (not shown). Furthermore, plasmid isolation fromtransduced MRSA strains demonstrated recovery of the pGW80A0001 plasmidas confirmed via sequencing of the isolated plasmid. The transductionresults thus demonstrated that the origin of replication of the reporterplasmid exhibits activity on all of the MRSA isolates tested.

FIG. 22 illustrates the luminescence measured from 80 clinical isolatesof MRSA and 28 clinical isolates of methicillin sensitive S. aureus(MSSA) transduced with the transduction particle. In the experiment,cultures of MRSA and MSSA were grown to an optical density at 600 nm of0.1 and then 100 uL of the cultures grown in Modified TSB were mixedwith 10 uL of GW24 cell lysates containing transduction particles andfurther incubated at 37° C. for a period of 4 hours prior to assayingfor luminescence. Luminescence measurements were conducted by adding 10uL of a 1 mM solution of Decanal, an aldehyde that triggers aluminescent reaction within cells expressing bacterial luciferase. Asexpected, luminescence was observed from both MRSA and MSSA transducedwith the S. aureus-specific non-replicative transduction particle.Furthermore, when cefoxitin was added to the cell cultures at the sametime as the addition of transduction particles, luminescence wasobserved from MRSA but not from MSSA, thus demonstrating the ability forthe transduction particles to report on both the presence of MSSA and ofMRSA. The luminescence results thus demonstrate that the promoterdriving luxAB expression exhibits activity on all of the S. aureusisolates tested.

Optimization of Non-Replicative Transduction Particle-Based Viable CellReporter MRSA Assay—Transduction Particle Reagent Formulation

The production and formulation of the non-replicative transductionparticle reagent was optimized to a final formulation. In summary, a 15L scale fermentation was performed using TSB media including peroxideinduction of GW24. The 15 L fermenter batch was inoculated from a 200 mLovernight seed culture (an inoculum ratio of 1.3% (v/v)). The culturewas induced at an O.D. of 0.8 with hydrogen peroxide and cooled to 25°C. post induction without pH or DO control. Culture supernatant washarvested by tangential flow filtration (TFF) the following morning forthe purpose of clarifying the phage transduction particles from the celldebris. The material was then further concentrated and diafiltered intoSM Buffer without gelatin and stored at 2-8° C. prior to final sterilefiltration and storage.

A detailed summary of the process is outlined below:

Seed flask growth (1) Inoculate 200 mL of TSB containing 5 ug/mLtetracycline with GW24 (2) Incubate at 37° C., 200 RPM for 10-18 hours.Fermentation Innoculation (15 L TSB with 5 ug/mL tetracycline) (1)Prepare the fermenter skid with the following fermentation conditions:37° C., agitation at 250 RPM, airflow at 15 LPM, and backpressure at 3psig. (2) Inoculate the fermentor using the 200 mL overnight seedculture. Induce Culture (1) Once the OD600 nm reaches 0.8 (0.6-0.9),induce the culture with 0.5 mM H2O2 (2) Increase temperature setpoint offermenter to 42° C. Post-Induction Conditions and Monitoring (1) Once 30minute induction is complete, reset temperature target for fermenter to25° C. (2) One hour post cooling, turn off air feed to fermenter and setagitation to zero (3) Monitor the fermentation culture at hourintervals, or more frequently as necessary, until the OD600 nm hasdecreased to or below 0.40. Harvest/Clarification (1) After thefermentation culture OD600 has reached a minimum less than or equal to0.40, take a 20 mL aseptic sample and add 30 μL of Benzonase to thefermentor. (2) Reset agitation to 250 rpm. Allow 60 minutes withagitation for Benzonase incubation. (3) Clarify the EOF sample with a 15minute centrifugation at 3000 g. (4) Pass the clarified material througha 0.45 uM membrane filter Concentration and Buffer Exchange (1)Concentrate the clarified culture by TFF using 500 kDa flat sheetmembrane 10-fold. (2) Diafilter the concentrated culture at a constantvolume against SM Buffer without gelatin using the 500 kDa TFF membraneused for concentration Final Filtration (1) Filter the concentratedbuffer exchanged material through a 0.2 μm filter. (2) Store the finalfiltered phage material at 2-8° C.

Various other reagents and formulations can be used as known to those ofskill in the art to derive the formulation.

Optimization of Non-Replicative Transduction Particle-Based Viable CellReporter MRSA Assay—Growth Media Formulation

A growth media formulation was optimized for the NRTP-based viable cellreporter MRSA assay. In order to produce luminescence in the NRTP-basedMRSA Assay, the media needs to be balanced for Staphylococcus aureusgrowth and have adequate concentration of cations and additives to favorNRTP transduction. The TSBmod media used in assays prior to thisdevelopment study was known to have precipitation issues that wouldaffect the stability of the media. Growth media formulation requiredstability in the final formulation with a goal of 1 year at roomtemperature.

Methods/Procedures: Cells Preparation for MRSA Assay

(1) Ten unique strains of MRSA for the Subset Assay and one uniquestrains of MSSA were tested in the MRSA Assay. (2) Overnight cultureswere started in a deep 96 well plate at a 1:50 dilution in TSB from afrozen one-time use stock and incubated at 37° C. on an orbital shakerfor >15 hours. MRSA/MSSA (8 μl) in TSB (392 μl) (3) The next day, a dayculture at a 1:50 dilution from the overnight culture was started in TSBin 96 well deep well plate (392 μl TSB + 8 μl cells) and incubated at37° C. on an orbital shaker for 4 hours. (4) Cells were spun in acentrifuge for 5 minutes at 1800 g force and 10° C., spent media wasaspirated without disturbing the pellet. (5) Spun cells were washed in50 mM Tris-HCl pH 7.2, centrifuged, buffer aspirated without disturbingpellet and re-suspended in 400 μl RPMI. RPMI is used in order to reducevariability in the metabolic state of cells and to mimic low metabolismas found in clinical samples. (6) Plate was covered with airpore sealand incubated on bench for 48 hrs. (7) OD was read by transferring 200μl of RPMI culture in a shallow well OD plate and blank well with RPMImedia alone was used to subtract blank OD. (8) Cells were normalized toOD 0.1 in 100 μl (9) Another dilution was made 1:10 in RPMI to yield anOD of 0.005

Assay base media was prepared to be tested as shown in Table 2 and arepresentative set of media modifications in preparation for MRSA assayare shown in Table 3.

TABLE 2 Base Media for Growth Media Formulation Development ComponentsTSB B2 BSS-2 Notes Enzymatic Digest of Soybean 3 0 3 Adjust pH to Meal(g) 7.2 with 10N Enzymatic Digest of Casein (g) 17 10 10 NaOH. YeastExtract (g) N/A 25 25 Autoclave or Sodium Chloride (g) 5 25 25 filtersterilize Dipotassium Phosphate (g) 2.5 1 0 alpha-D Glucose (g) 2.5 5 5Volume (litre) 1 1 1

TABLE 3 Base Media Modifications for Growth Media FormulationDevelopment Concentration of salt/additives for modification BaseTris-HCl media Mod CaCl2 MgCl2 BGP pH7.0 EDTA HEPES (30 ml) number (mM)(mM) (mM) (mM) (mM) (mM) B2 M53 5.0 2.0 0.0 50.0 10.0 0.0 BSS-2 M50 10.02.0 60.0 50.0 10.0 0.0 BSS-2 M54 6.7 3.3 60.0 50.0 0.0 0.0 BSS-2 M55 5.05.0 60.0 50.0 0.0 0.0 BSS-2 M56 6.7 3.3 60.0 0.0 0.0 10.0 BSS-2 M57 5.05.0 60.0 0.0 0.0 10.0 TSB M1  5.0 10.0 60.0 0.0 0.0 0.0 (original) TSBM58 5.0 10.0 60.0 0.0 11.1 0.0

To each media preparation, NRTP and Cefoxitin was added according toTable 4 below to make the NRTP media reagent:

TABLE 4 MRSA Assay Growth Media/Transduction particle reagentcombination Final 30 ml media Concentration Cefoxitin 5 ug/ml GW24Lysate 30X

The MRSA Assay was run with the following steps:

(1) Assay Plate Setup: Add 198 μl of Phage Media Reagent and 2.0 μl ofeach dilution of bacteria 0.05 OD and 0.005 OD in RPMI (roughlyequivalent to 20,000 and 2,000 CFU/mL, respectively) or 2.0 uL of RPMIas a blank. (2) Incubate Assay Plate: Incubate Assay plate on orbitalshaker at ~100 rpm for 4 hours at 37° C. (3) Prepare Luminometer(Molecular Devices SpectraMax L): Wash reagent line with 70% ethanolfollowed by DI water then prime with the substrate reagent. Set upsoftware as Fast Kinetic with injection of 50 μL of substrate reagent at250 μl/sec after 10 baseline points and read at 40 points every 0.25seconds. (4) Run Assay: Test each bacterial dilution plate, afterletting plate equilibrate to room temperature for 5 minutes.

Analysis

(1) Determine cutoff by averaging blank RLU across all replicates andtime points and adding three standard deviations. (2) Determine maximumRLU for each sample using SoftMaxPro. (3) Determine if the maximum RLUwas greater than the cutoff RLU, and if so, then the sample data wasused for comparisons of media performance. (4) Normalize all max RLUvalues to the Max RLU in TSB M1 (media in use until development started)for the strain being analyzed at the specific dilution. (5) Average thenormalized RLU values across all MRSA strains for a particular media andits modification (6) Average the averages for the two dilution plates,ultimately leading to a single numerical value representing the foldincrease in performance based on RLU of a particular media across 10different MRSA strains in 2 cell dilutions tested.

Results of NRTP-Based Viable Cell Reporter MRSA Assay

Determination of Cutoff RLU: The average and standard deviation of theRLU was calculated across all time points (25) for each blank replicate(4). The cutoff was calculated for each plate as the average blank RLUplus three standard deviations.

Determination of Relative Improvements: The maximum RLU was exported foreach sample (blanks, MSSA and MRSA at all dilutions) from SoftMaxPro andcompared to the cutoff RLU. If the sample had 2 data points greater thanthe cutoff for phage concentration, then the max RLU value was utilizedfor analysis.

The values were normalized by dividing a particular max RLU by the maxRLU of its control condition (that strain in TSB M1-origianl media, atthe dilution being analyzed). The ratios obtained were averaged across10 MRSA for each media condition and each dilution, as shown in Table 5.The average across the two dilutions is also shown in the table.

TABLE 5 MRSA Assay Results from Various Growth Media FormulationsAverage for Media Plate 1 Plate 2 both dilutions B2 M53 1.89 1.88 1.89BSS-2 M50 1.37 1.47 1.42 BSS-2 M54 1.50 1.76 1.63 BSS-2 M55 1.82 2.902.36 BSS-2 M56 2.38 6.00 4.19 BSS-2 M57 2.00 3.92 2.96 TSB M1 1.00 1.001.00 TSB M58 1.18 0.96 1.07

Conclusions

BSS2-M56 exhibited the best performance on average across the variousmedia tested. HEPES buffer based media performed better than Tris-HClbuffered media. HEPES is known to be a biologically favorable bufferingsystem as opposed to Tris-HCl. B2 based base/broth had betterperformance than TSB based broth.

Various other reagents and formulations can be used as known to those ofskill in the art to derive the formulation. Other suitable formulationswere developed via similar experiments as described above. Examples ofother suitable formulations are included below in Tables 6, 7, and 8.

TABLE 6 BSC Media Formulation BSC Components Amount Enzymatic 14.5 gDigest of Casein Yeast Extract 35.5 g Sodium 35.5 g Chloride alpha-DGlucose   7 g Total Vol   1 L

TABLE 7 BSC Media Modification BSC-M64 Chemical Name Final (Assay) ConcBGP (mM) 60.0 HEPES (mM) 10.0 LiCl (mM) 84.0 BSC To 1 L

TABLE 8 Transduction Particle Media Modification Transduction ParticleFormulation (PM4) Chemicals Final (Assay) conc CaCl2 (M) 0.00667 MgCl2(M) 0.00335 HEPES (M) 0.01000 GW24 lysate stock 0.01250 Sodium Azide (%)0.0006 Water To 1 mL

Optimization of Non-Replicative Transduction Particle-Based Viable CellReporter MRSA Assay—Substrate Reagent Formulation

In order to produce luminescence in the MRSA Assay, the SubstrateReagent must include an aldehyde as a substrate for luciferase. Aninitially developed aliphatic aldehyde formulation (4.2 mM Tridecanal inTSB) was not stable and formed a heterogeneous emulsion rather than asolution. This example outlines the development of a Substrate Reagentformulation that addresses these issues with a goal of 6 months at roomtemperature or 2-8° C. stability.

This example describes the steps that were taken to develop theSubstrate Reagent to a final formulation.

Methods/Procedures

All screening and stability experiments were tested using a “ModelSystem” that consists of S. aureus strain RN4220 harboring aLuxAB-expressing plasmid. The typical preparation and testing method wasas follows.

(1) Overnight Culture: 2 mL TSB + 1 uL of 10 mg/mL Tetracycline + 1colony of Model System Bacteria from TSA plate, shaking at 225 rpmovernight at 37° C. (2) Day Culture: Diluted overnight culture 1:50 or1:100 into TSB + 5 ug/mL Tetracycline, shaking at 225 rpm for 1.5-2hours at 37° C. (3) Normalize Day Culture: Measured 1 mL of day cultureon Nanodrop with cuvette at 600 nm, blanking with TSB + 5 ug/mLTetracycline. Diluted to 0.1 OD with TSB + 5 ug/mL Tetracycline. (4)Dilute Culture for Testing: Diluted 0.1 OD Culture with TSB + 5 ug/mLTetracycline to a 1:200, 1:2000 and 1:20000 dilution which was roughlyequivalent to 100000, 10000 and 1000 CFU/mL. (5) Plate Bacteria: Added200 uL of each dilution and a blank (TSB + 5 ug/mL Tetracycline with nobacteria) in three replicates to a Greiner Bio-one white assay plate foreach substrate to be tested. (6) Prepare Luminometer (SpectraMax L):Wash reagent line with 70% ethanol followed by DI water then prime withthe substrate. Set up software as Fast Kinetic with injection of 50 uLsubstrate at 250 ul/sec after 10 baseline points and read at 40 pointsevery 0.25 seconds. (7) Run Assay: Test each formulation of SubstrateReagents with washing and priming SpectraMax L between each substrate.Bring all Substrate Reagents to room temperature before testing.

All confirmation experiments were tested using the MRSA Assay in orderto ensure similar results on the actual assay as the Model System usedto screen new formulations.

(1) Prepare Culture: Ten MRSA low performing strains and one MSSA strainwere grown to log-phase in TSB in a 2 mL deep well block. Cells werespun down, washed with 1 × PBS then resuspended in RPMI media. (2)Normalize Bacteria: Measure 200 uL of RPMI culture and RPMI blank inGreiner Bio-one clear plate on VersaMax at 600 nm. Subtract blank ODfrom each strain. Normalize each strain to 0.05 OD in RPMI media. (3)Dilute Bacteria: Dilute 0.05 OD culture 1:10 in RPMI media to 0.005 OD.(4) Prepare Phage Media Reagent: Add Phage, Cefoxitin and SodiumPyruvate to BSS-M56 including: a. Cefoxitin (5 ug/mL) b. GW24 lysatestock (0.03X) c. Sodium Pyruvate (0.025M) (5) Set up Assay Plate = Add198 uL of Phage Media Reagent and 2 uL of each dilution of bacteria(0.05 OD and 0.005 OD in RPMI, roughly equivalent to 20000 or 2000CFU/mL) or 2 uL of RPMI as a blank in two replicates. (6) Incubate AssayPlate = Incubate Assay plate on orbital shaker at ~100 rpm (speed 3) for4 hours at 37° C. (7) Prepare Luminometer (SpectraMax L) = Wash reagentline with 70% ethanol followed by DI water then prime with thesubstrate. Set up software as Fast Kinetic with injection of 50 uLsubstrate at 250 ul/sec after 10 baseline points and read at 40 pointsevery 0.25 seconds. (8) Run Assay = Test each formulation of SubstrateReagents with washing and priming SpectraMax L between each substrate.

Experiments for the development of Substrate Reagent formulation weredesigned to improve the following:

(1) Improve solubility via adding surfactants (Tween 20, Triton X-100,NP-40, Brij-35, SNS, etc.), adding solvents (Ethanol, Methanol, DMSO,etc.), adding non-volatile oils (Castor Oil) (2) Improve stability viaadding stabilizers (Triethanolamine, Cyclodextrin etc.), addingantioxidants (Vitamin E, Vitamin E Acetate, Vitamin E PEG 1000, Oxyrase,etc.), adjust method of Tridecanal addition (with surfactant, withsolvent, into final solution, with antioxidant, etc.), storingTridecanal and Substrate Reagent under nitrogen to reduce oxidation ofaldehyde, and reducing possibility of microbial contamination by addingpreservatives such as ProClin and by sterile filtration of the SubstrateReagent. (3) Improve Assay Performance via adjustment of the pH of theformulation and the pH buffer system (4) Improve overall performance viadetermining the aldehyde with highest RLU output (tested aldehydes from6-14 carbons in multiple formulations to determine if an improvement insolubility, stability and assay performance was observed). (5) Improveoverall performance via adding antifoam in order to reduce foamingduring preparation of reagent and addition of reagent to sample duringthe assay.

Analysis and Results

The kinetic reaction was plotted for each sample and a line fit to theaverage at each read point of three replicates. Typically results showedat 1:2000 dilution of 0.1 OD model system bacteria, roughly equivalentto 10,000 CFU/mL or 2,000 CFU/assay.

The normalized maximum RLU to that of the reference substrate reagentwas analyzed for stability experiments. At each stability time point,maximum RLU for each sample was normalized to the reference substratemaximum RLU. Normalized Maximum RLU was plotted over time points andlinear regression with 95% CI was plotted.

Conclusions

The key parameters adjusted from the reference formulation for producinga final Substrate Reagent formulation are summarized in Table 9.

TABLE 9 Summary of Reagent Formulation Development Results Modificationto Substrate Reagent Reason 4.2 mM Tridecanal + TSB Original SubstrateReagent Remove TSB Reduce possibility of contamination Add 1% Tween 20Improve Solubility Adjust to pH3 with 79.45% 0.1 M Improve AssayPerformance Citric Acid-19.55% 0.2 M Sodium Phosphate Dibasic Buffer AddTridecanal directly to Improve Stability concentrated surfactant AddFiltering of Substrate Reagent Improve Stability through 0.2 um PESmembrane Add 0.05% ProClin 300 Improve Stability Add TriethanolamineImprove Stability Change 1% Tween 20 to 0.5% Triton Improve Stability,Improve X-100 Solubility Change from 79.45% 0.1 M Citric Improve AssayPerformance, Acid-19.55% 0.2 M Sodium Phosphate reduce possibility ofprecipitation Dibasic Buffer to 82% 0.1 M Citric with removal ofphosphate buffer Acid-18% 0.1 M Sodium Citrate Buffer, remain at pH 3Add 100 ppm Antifoam Y30 Improve Assay Performance Add 0.5% Vitamin EAcetate Improve Stability, reduce precipitation Change PrimaryTridecanal Improve Assay Performance Manufacturer from Alfa Aesar toSigma/OmegaChem Change 0.5% Vitamin E Acetate to 1- Improve AssayPerformance, 2% Vitamin E PEG 1000 Improve Solubility, Improve Stability

Two Substrate Reagent Formulations were prepared for two differentstorage temperatures, one for storage at 2-8° C. and one at 18-24° C.

Final Substrate Reagent Formulations Stored at 2-8° C.

Formulation: 0.5% Triton X−100+4.2 mM Tridecanal+0.5% Vitamin EAcetate+100 ppm Antifoam Y30+0.5% Triethanolamine+82% 0.1 M CitricAcid+18% 0.1 M Sodium Citrate @pH3+0.05% ProClin 300. The formulationdid not precipitate after 1 month at 2-8° C. and was able to detect MRSAstrains the same as on Day 0.

Final Substrate Reagent Formulations Stored at 18-24° C.

Formulation: 0.5% Triton X−100+6.3 mM Tridecanal+100 ppm AntifoamY30+0.5% Triethanolamine+82% 0.1 M Citric Acid+18% 0.1 M Sodium Citrate@pH3+2% a-Tocopherol-PEG 1000 Succinate+0.05% ProClin 300. Theformulation did not precipitate after 1 month at 18-24° C. and was ableto detect MRSA strains the same as on Day 0.

Various other reagents and formulations can be used as known to those ofskill in the art to derive the formulation.

Analytical Performance of Non-Replicative Transduction Particle-BasedViable Cell Reporter MRSA Assay

The analytical performance of the optimized NRTP MRSA assay wasexamined, including an analysis of the assay's limit of detection and ananalysis of the cross-reactivity and microbial interference of the assaywhen challenged with non-target organisms.

A) Limit of Detection Assay

The Limit of Detection of the NRTP assay was assessed via determiningthe lowest amount of MRSA cells representing various strains that couldproduce a relative light unit (RLU) signal above that of a thresholddetermined from blank samples. MRSA strains included the SCCmec Types I,II, and IV as well as a MRSA strain carrying the mecA gene variantmecC—a strain of MRSA that conventional FDA-cleared MRSA PCR assays havefailed to detect.

The following key materials were used in the clinical performance study:

Growth Media Reagent: BSS-M56

Substrate Reagent: Final Substrate Reagent Formulations to be stored at18-24° C. as described above.

Transduction Particle Reagent: BSS-M56 base with 10 ug/mL (i.e. 2×concentration) cefoxitin and transduction particle reagent as describedabove at 2× concentration.

LoD Study Protocol:

Overnight Culture:

For each MRSA strain and a MSSA negative control strain, 2 mL of TSBwere inoculated with a colony of the strain previously grown on TSAplates. Overnight MRSA cultures included 5 ug/mL cefoxitin. All sampleswere incubated overnight at 37° C. in a shaking incubator.

Day Culture:

20 uL of each of the overnight cultures were transferred into a newculture tube containing 2 mL of Growth Media Reagent. The inoculums werethen incubated at 37 C with shaking for approximately 1 hr 45 min, untilthe OD (600 nm) reached 0.1.

Serial Dilutions:

-   -   a) 1000 uL of each of the samples were dispensed into row A of 2        mL deep well 96-well plate.    -   b) The remaining rows (B-H) were then filled with 900 uL of        Growth Media Reagent.    -   c) 10-fold serial dilutions were then prepared taking 100 uL        from row A and mixing in row B, etc., such that row H contained        samples of row A material at 10⁻⁷ dilution.

Enumeration of Bacterial Load:

5 uL of each well of row E was spotted onto a TSA plate which was thentilted to allow the spot of liquid to spread onto the plate (in order tolater facilitate colony counting). (Row E is a 10⁻⁴ dilution of row A).Plates were then incubated overnight at 37° C.

Assay Preparation:

a) Wells of a white 96 well assay plate were filled with 100 uL of 2×Transduction Particle Reagent.

b) Rows F and G (i.e., 10⁻⁵ and 10⁻⁶-fold dilutions of row A,respectively) were then used to fill wells of the 96 well assay platecontaining Transduction Particle Reagent such that each sample was addedto the plate in four-replicates.

c) The plate was then sealed with a breathable seal and incubated for 4hours at 37° C. with moderate shaking, 50 rpm.

At the end of 4 hours, the plate was remove from incubator andimmediately measured for luminescence on a SpectraMax L that injected 50μl of the Substrate Reagent and measured luminescence for a period of 1minute.

Analysis:

The luminescence data from each sample was plotted as RLU vs. time.Blank samples were used to determine a Cutoff calculated from all timepoints of the blank samples using the following formula: (Mean BlankRLU+3*SD Blank RLU)

The average peak RLU post-substrate injection was then obtained for eachsample in order to determine the sample of highest dilution for which anRLU value was generated that was above the blank samples Cutoff. Thecolony forming unit (CFU) counts at the highest dilution for which anRLU value was generated that was above the blank samples Cutoff wasdetermined from the enumeration study, and this CFU count was reportedas the LoD in the study.

Results:

The LoD for all MRSA samples tested was determined to be below 10 CFU.Table 11 summarizes the results of the lowest LoDs obtained in thestudy.

TABLE 11 Results of the lowest LoDs obtained in the LoD study. SCCmecLoD Type (CFU) I 3 II 2 IV 3 mecC 1

All MRSA strains tested resulted in fewer than 10 CFU detected with theNRTP assay above a Cutoff calculated from blank samples. MSSA did notgenerate RLU values above the blank samples Cutoff

RLU values are shown at the highest dilution for which an RLU value wasgenerated that was above the blank samples Cutoff were plotted as theaverage RLU value and standard deviation for the four replicates testedfor each sample. The horizontal axis is set at the blank samples Cutoffand the CFU counts for the sample that generated each RLU data point issuperimposed with the data. All MRSA samples generated RLU values abovethe Cutoff while MSSA did not.

Cross-Reactivity and Microbial Interference Study

A cross-reactivity and microbial interference study was performed. Thepurpose of the study was to test a set of bacterial strains commonlyencountered in clinical samples and known to potentially be in the hostrange of the bacteriophage φ80α in the MRSA Assay to see if there wascross reactivity or interference of these strains with phage orsubstrate used in the test.

Previous experiments with clinical samples had resulted in falsepositive results with a presence of Enterococci faecalis andStaphylococcus epidermidis as indicated from the presence of blue andwhite colonies when plating on BBL™ CHROMagar™ Staph aureus plates. Inaddition, Listeria monocytogenes and Listeria innocua may be within theinfective or penetrative host range of the phage φ80α which may alsocontribute to cross-reactivity in the MRSA assay. The study testedEnterococci faecalis, Staphylococcus epidermidis, Listeria monocytogenesand Listeria innocua for cross reactivity/interference with ViabilityMRSA assay. Each strain was tested at high cell numbers in the order of10⁶, 10⁷ or 10⁸ cells in the assay volume. Tests were done without theaddition of GW24 lysate to address potential autoluminescence ofstrains.

Experiment 1 tested various strains (MSSA-S121, NRS# 9-Staphylococcushaemolyticus, NRS# 6-Staphylococcus epidermidis, ATCC12228-Staphylococcus epidermidis, ATCC 15305-Staphylococcussaprophyticus, ATCC 29212-Enterococcus. faecalis, ATCC 60193-Candidaalbicans, ATCC 12453-Proteus mirabilis) for luminescence at high cellnumbers under normal assay conditions.

Experiment 2: A subset of strains that were luminescent from Experiment1 were re-assayed in the presence of various antibiotics at variousconcentrations to quench background luminescence.

Experiment 3: E. faecalis and S32 (MRSA) were tested with varioussubstrate formulations developed as described above without GW24 lysateand without incubation.

Experiment 4: ATCC 33090-Listeria innocua and ATCC 19111-Listeriamonocytogenes were tested for background signal and non-specificluminescence and retested with various substrate formulations developedas described above along with E. faecalis and S. epidermidis.

Experiment 5: E. faecalis was retested with a final substrateformulation developed as described above.

Substrate Reagent formulations tested in this study are summarized inTable 9.

TABLE 10 Substrate Reagent Formulations Experiment Substrate Description1 Original Substrate 1% Tween20 + 4.2 mM Tridecanal, 2 OriginalSubstrate pH 3.0 3 Substrate 1 6.3 mM Tridecanal + 0.5% Vitamin EAcetate, pH 3.0 Substrate 2 20 mM Nonanal + 0.5% Vitamin E Acetate, pH3.0 Substrate 3 8.4 mM Tridecanal + 0.5% Vitamin E Acetate, pH 3.0Substrate 4 6.3 mM Tridecanal + 1% a-Tocopherol- PEG 1000 Succinate, pH3.0 4 Original substrate 1% Tween20 + 4.2 mM Tridecanal, pH 3.0Substrate 5 0.5% Triton + 4.2 mM Tridecanal (Sigma) + 0.5% Vitamin EAcetate, pH 3.0 5 Substrate 6 6.3 mM Tridecanal + 2% VitE PEG, pH 3.0

Methods/Procedures:

The following were steps performed for the MRSA Assay.

A) Strains Grown for Experiments 1-5

On the day before the assay, an overnight culture was started in a deep96 well plate at a 1:50 dilution in TSB from a frozen one-time use stockand incubated at 37° C. on an orbital shaker for >15 hours. Bacteria (8uL) in TSB (392 uL).

The absorbance of culture was measured on Versamax. The TSB was set asblank in template on SoftmaxPro. Optical density (OD) was measured at600 nm.

On the day of the assay, cells were re-suspended to an OD 0.5 to set upthe assays. Prepared BSS-M56 for Experiments 1-5.

B) Transduction Particle Media Reagent was Prepared for all Experiments1, 2, 4 and 5 (No Transduction Particle Reagent Used in Experiment 3):15 ug/mL Cefoxitin+GW24 Lysate Stock from as Described Above at 30×.

C) Sample Preparation:

Various dilutions were made from overnight cultures of strains. Allstrains were diluted BSS M56.

D) MRSA Assay was Run for Experiments 1-5

Media was loaded with or without phage and cefoxitin at 5 μg/ml to assayplate. 2.5 ul cells were added. The assay plate was incubated with aplate lid at 37° C. on an orbital shaker with the speed set toapproximately 100 rpm for 4 hours.

Next, the assay plates were measured on the SpectraMax L with thefollowing standard assay parameters:

Fast Kinetic Luminescence

Read for 20 time points at 0.5 second intervals. Substrate was injectedwith M injector with 50 uL/well at 250 ul/sec including 5 baselinereads. No incubation temperature was set and was read at roomtemperature.

The SpectraMax L was primed with Substrate Reagent before running theassay.

The results were analyzed with the following:

A) Determined cutoff by averaging blank RLU across all replicates andtime points and adding three standard deviations.

B) Determined maximum RLU for each sample using SoftMaxPro.

C) Determined if the maximum RLU was greater than the cutoff RLU, and ifso, then the sample data was used for analysis.

Results Summary Experiment 1

Various strains were tested for cross reactivity and interference usingthe Original Substrate formulation, out of those tested, NRS#9-S.haemolyticus, NRS #6-S. epidermidis and E. faecalis tested falsepositive in MRSA assay.

Experiment 2

Out of the three strains tested, NRS #9 and E. faecalis tested MRSApositive with all Cefoxitin conditions tested. All three strains (NRS#9, E. faecalis, NRS #6) tested positive when no transduction particlereagent was used in the assay, indicating that non-specific luminescencewas not transduction particle reagent-dependent but rather strain andsubstrate reagent dependent. Carb (Carbencillin) at all concentrationstested was effective in removing the false positive signal.

Experiment 3

E. faecalis gave a positive signal without transduction particlereagent. MRSA strain S32 also gave a positive signal withouttransduction particle reagent. This result was indicative of thesubstrate reagent causing background luminescence. Substrate 4 waseffective in eliminating background signal in the assay.

Experiment 4

Strains ATCC 33090-Listeria innocua, ATCC 19111-Listeria monocytogenes,were tested for luminescence with transduction particle reagent andsubstrate reagent as Listeria sp. can be within the host range of thebacteriophage used in the MRSA assay. Luminescence was observed from L.innocua with and without transduction particle reagent using OriginalSubstrate formulation indicating that the luminescence was due tonon-specific reaction potentially with the substrate. Substrate 5 waseffective in eliminating luminescence from Listeria but not E. faecalis.

Experiment 5

Retested E. faecalis with Substrate 6. In two independent runs on twodifferent days with high load of cells at 0.5 OD, the assay yieldednegative results.

Conclusions

The cross-reactivity study demonstrated background luminescence fromseveral bacterial species at high loads. The light output did notrequire transduction particle reagent and certain substrate formulationsutilizing phosphate ions contributed to non-specific signal. Because nolight output from cross-reactive species was observed from the use oftransduction particle reagent, in the case that φ80α penetratescross-reactive species, light output is prevented from the lack ofactivity of the S. aureus PclpB promoter that is operatively linked tothe bacterial luciferase genes and/or the lack of activity of the S.aureus pT181 origin of replication within these species.

Replacing the buffer from sodium phosphate dibasic in the formulationwith sodium citrate and citric acid eliminated background luminescencefrom all cross-reactive species tested except for E. faecalis. Substrate6 with the added ingredient of a tocopherol-PEG 1000 Succinateeliminated the remaining non-specific signal from E. faecalis.

Clinical Performance of Non-Replicative Transduction Particle-BasedViable Cell Reporter MRSA Assay—Results with Reference to Direct Platingonto CHROMAgar MRSA II

A MRSA screening assay was developed employing φ80α-based luxABexpressing non-replicative transduction particles (NRTP). The assayconsisted of adding NRTP to a clinical sample suspected of containingMRSA, incubating the sample for a period of 4 hours at 37° C., and thenassaying the incubated sample by injecting an aldehyde into the samplewhile measuring for luminescence with a photomultiplier tube. Theresults of the assay were compared to that of commercially availablechromogenic media designed for the detection of MRSA as a reference inorder to determine the sensitivity and specificity of the assay. TheNRTP-based assay was expected to correlate well with the culture-basedreference since both require the presence of viable MRSA cells and bothrely on the expression of the MRSA phenotype. The results showedexcellent correlation with the reference.

The purpose of the study was to determine the performance of theNRTP-based MRSA Assay with reference to CHROMAgar MRSA II from testingremnant nasal swab samples collected for the purpose of MRSA screening.

Scope:

De-identified nasal swab samples collected from patients for the purposeof MRSA surveillance by a clinical institution were tested for thepresence of MRSA using the NRTP-based MRSA Assay, CHROMAgar MRSA II,CHROMAgar SA and Blood Agar TSA via direct plating and via enrichedculture followed by plating. The results of the NRTP-based MRSA Assaywere compared with the results of the CHROMAgar MRSA II assay in orderto calculate the sensitivity and specificity of the NRTP-based MRSAAssay with reference to CHROMAgar MRSA II.

The following key materials were used in the clinical performance study:

Growth Media Reagent: BSS-M56

Substrate Reagent: Final Substrate Reagent Formulations to be stored at18-24° C. as described as described above

Transduction Particle Reagent: BSS-M56 base with 10 ug/mL (i.e. 2×concentration) cefoxitin and transduction particle reagent as describedabove at 2× concentration.

Methods/Procedures

Clinical Sample Description:

Sample transport tubes containing liquid Amies (220093-BD BBL™CultureSwab™ Liquid Amies) were provided to a clinical institution forcollecting de-identified remnant nasal swabs collected by the clinicalinstitution. Prior to placing the nasal swabs into the provided sampletransport tube, the clinical institution used the swab for performingtheir own direct culture MRSA screening by streaking the swab onto aculture plate. More specifically, anterior nares specimens werecollected at the clinical institution internal standard procedures andusing the clinical institution's standard collection swab. The clinicalinstitution then performed direct culture screening with the swab. Theremnant swab was then added to the sample transport tube in which theswab tip was submerged in the Amies buffer in the sample transport tube.Samples were then kept at room temperature for 2-24 hours prior tofurther processing.

Sample Handling:

Upon receipt, samples were stored overnight at room temperature in abiosafety cabinet upright to ensure swab immersion in the sampletransport tube Amies buffer. After overnight storage, samples werefurther processed as follows.

Clinical Sample Preparation

Using a 1 mL Pipette, 300 μl of Growth Media Reagent was added to 15 mLfalcon tubes.

The swabs from remnant nasal swabs were removed from the originaltransport tube and immersed into the Growth Media Reagent in acorresponding falcon tube. The swab contents were then eluted into theGrowth Media Reagent in the falcon tube by rolling it back and forth inthe Growth Media Reagent 4-6 times. The swab was then placed back intothe original transport tube and stored at 2-8° C. until the end of thestudy while the eluted clinical samples in the falcon tube weretransferred to 1.5 mL tubes and kept at room temperature until furtherprocessing.

Running the NRTP MRSA Assay:

The following samples were loaded directly into a white 96 well assayplate.

Clinical Samples: 100 μl of the eluted material of each clinical samplein singlet.

MRSA positive control: 2 μl of a thoroughly mixed 0.1 OD culture of aknown MRSA isolate into 98 uL of Growth Media Reagent in triplicate.

MSSA negative control: 2 μl of a thoroughly mixed 0.1 OD culture of aknown MSSA isolate into 98 uL of Growth Media Reagent in triplicate.

Blanks: 100 μl of Growth Media Reagent in triplicate.

To each sample, 100 μl of Transduction Particle Reagent was added. Theassay plate was then placed in an incubator set at 37° C., shaking onorbital shaker for 4 hours. At the end of 4 hours, the plate was removedfrom incubator and immediately measured for luminescence on a SpectraMaxL that injected 50 μl of the Substrate Reagent and measured luminescencefor a period of 1 minute.

Bacteria Plating for Clinical Sample CFU Enumeration:

Each eluted clinical sample was plated in order to determine bacterialcolony counts on CHROMAgar MRSA II, CHROMAgar SA and Blood Agar (TSA II)via direct and enriched culture as follows. Organism CFU counts weredetermined by direct plating. MRSA CFU counts were determined by platingon CHROMAgar MRSA II. S. aureus CFU counts were determined by plating onCHROMAgar SA plate. CFU counts of any organism whose growth is supportedby Blood Agar TSA were determined by plating on Blood Agar TSA. In thecase that direct plating did not produce colonies due to the load oforganisms being below the limit of detection of the plates used, sampleenrichment was also performed by incubating a portion of the elutedclinical sample in TSB overnight at 37° C. with shaking and then againplating the enriched culture on CHROMAgar MRSA II. All plates wereincubated for 20-24 hours at 37° C. After incubation, the CFU counts ofany colonies appearing on each plate were recorded.

Analysis:

The presence and CFU load of MRSA, S. aureus, and total organisms pereluted clinical sample were calculated based on the CFU counts obtainedon CHROMAgar MRSA II, CHROMAgar SA, and Blood Agar TSA, respectively.

NRTP Assay analysis:

Data from each sample were plotted as RLU vs. time.

Cutoff Determination:

The Assay Cutoff was calculated from all time points of the blanksamples using the following formula: (Mean Blank RLU+3*SD Blank RLU).

MRSA Positive Determination:

The RLU of each time point after substrate injection was determined tobe above or below the Assay Cutoff. If two or more data points afterinjection were above the Assay Cutoff then the sample was designated as“MRSA Positive.”

Results:

The MRSA positive results of the NRTP Assay were compared to those ofthe direct and enriched culture plating onto CHROMAgar MRSA II. Thefollowing calculations were conducted in order to determine the NTRPAssay Sensitivity and Specificity with reference to CHROMAgar MRSA II.

True Positive (TP) Sample that produced a MRSA positive result on boththe NRTP Assay and CHROMAgar MRSA II True Negative (TN) Sample thatproduced a MRSA negative result on both the NRTP Assay and CHROMAgarMRSA II False Positive (FP) Sample that produced a MRSA positive resulton the NRTP Assay and a MRSA negative result on CHROMAgar MRSA II FalseNegative (FN) Sample that produced a MRSA negative result on the NRTPAssay and a MRSA positive result on CHROMAgar MRSA II Sensitivity =TP/(TP + FN) Specificity = TN/(TN + FP)

Results with Reference to Direct Plating onto CHROMAgar MRSA II

Table 11 shows following results were obtained comparing the NRTP Assaywith reference to direct plating on CHROMAgar MRSA II.

TABLE 11 NRTP Assay Results vs. Direct Plating on CHROMAgar MRSA IIResults CHROMAgar CHROMAgar NRTP NRTP Total MRSA II MRSA II ASSAY ASSAYTrue True False False Samples Positive Negative Positive NegativePositive Negative Positive Negative 69 7 62 12 57 7 57 5 0

Based on the above data, the sensitivity and specificity of the assaywith reference to direct plating onto CHROMAgar MRSA II were calculatedto be:

Sensitivity=100%

Specificity=92%

Clinical Performance of Non-Replicative Transduction Particle-BasedViable Cell Reporter MRSA Assay—Results with Reference to EnrichedCulture, Followed by Plating onto CHROMAgar MRSA II

Based on the results with reference to direct plating on CHROMAgar MRSAII, all clinical samples were re-tested with reference to enrichedculture, followed by plating on CHROMAgar MRSA II. The rationale for thefollow-on testing was based on the possibility that false positiveresults when compared to direct plating may indeed be true positivesthat were detected by the NRTP assay but may have been missed by directplating. A portion of the remaining eluted swab samples were re-testedvia the NRTP assay as described above. Another portion of the remainingeluted swab samples were also tested via enriched culture, followed byplating onto CHROMAgar MRSA II. Enriched culture testing consisted ofadding 100 uL of the remaining eluted swab material to 2 mL of TSB andincubating at 37 C with shaking for a period of 18-24 hours. Theresulting culture was then streaked onto CHROMAgar MRSA II in order todetermine the presence of MRSA in the culture. Table 12 summarizes thedata from both the direct plating and enrichment followed by platingassays—only the samples that produced a MRSA positive result on eitherNRTP Assay or CHROMAgar MRSA II are shown.

TABLE 12 NRTP Assay Results vs. Direct Plating and Enriched Culturefollowed by Plating on CHROMAgar MRSA II Direct Enrichment + SampleCHROMagar Enrichment + CHROMagar # NRTP Assay MRSA II NRTP Asssay MRSAII 1 + + + + 2 + + + + 3 + + + + 4 + + + + 5 + + + + 6 + + + + 7 + + + +8 + − + + 9 + − + + 10 + − + + 11 + − + + 12 + − + −

Only the samples that produced a MRSA positive result on either NRTPAssay or CHROMAgar MRSA II are shown.

Table 13 shows following results were obtained comparing the NRTP Assaywith reference to enriched culture of clinical samples, followed byplating on CHROMAgar MRSA II.

TABLE 13 NRTP Assay Results vs. Enriched Culture Followed By Plating onCHROMAgar MRSA II Results CHROMAgar CHROMAgar NRTP NRTP Total MRSA IIMRSA II ASSAY ASSAY True True False False Samples Positive NegativePositive Negative Positive Negative Positive Negative 69 11 58 12 57 1157 1 0

Based on the above data, the sensitivity and specificity of the assaywith reference to enriched culture followed by plating onto CHROMAgarMRSA II was calculated to be:

Sensitivity=100%

Specificity=98.3%

Example 8: NRTP-Based Assay for Antimicrobial SusceptibilityTesting—Correlation of Minimum Inhibitory Concentration to LuminescenceOutput

In another example, a S. aureus cefoxitin susceptibility assay wasdeveloped to determine the minimum inhibitory concentration of cefoxitinrequired to inhibit the growth of cefoxitin resistant S. aureus. Unlikea MRSA cefoxitin resistance assay as described above, whichdifferentiates cefoxitin sensitive from cefoxitin resistant S. aureus,the MRSA cefoxitin susceptibility assay in this example describes thedevelopment of an assay to determine the minimum amount of cefoxitinneeded to inhibit the grown of S. aureus in the presence of cefoxitin.

The following key materials were used in the clinical performance study:

Growth Media Reagent: BSS-M56

Substrate Reagent: Final Substrate Reagent Formulations to be stored at18-24° C. as described in Example 7.

Transduction Particle Reagent: BSS-M56 base with 10 ug/mL (i.e. 2×concentration) cefoxitin and transduction particle reagent as describedin Example 7 at 2× concentration MIC Study Protocol.

Overnight Culture: For each MRSA strain (NRS35 and S7) and a MSSAnegative control strain (MSSA121), 2 mL of TSB were inoculated with acolony of the strain previously grown on TSA plates. Overnight MRSAcultures included 5 ug/mL cefoxitin. All samples were incubatedovernight at 37° C. in a shaking incubator.

Day Culture: 20 uL of each of the overnight cultures were transferredinto a new culture tube containing 2 mL of Growth Media Reagent. Theinoculums were then incubated at 37 C with shaking for approximately 1hr 45 min, until the OD (600 nm) reached 0.1.

MIC Determination Via Plating:

a) Each of the day cultures was streaked onto TSA plates containingcefoxitin at 4, 8, 16, 32, 64, and 128 ug/mL.

b) Plates were incubated for 18 hours at 37 C to determine growth.

NRTP Assay Preparation:

a) Wells of a white 96 well assay plate were filled with 100 uL of 2×Transduction Particle Reagent.

b) For each of the day cultures, five wells were then filled with 100 uLof day culture.

c) For each of the day cultures, cefoxitin was added to one well eachsuch that the cefoxitin concentration in the well was at 4, 8, 16, 32,64, and 128 ug/mL.

d) The plate was then sealed with a breathable seal and incubated for 4hours at 37° C. with moderate shaking, 50 rpm.

At the end of 4 hours, the plate was remove from incubator andimmediately measured for luminescence on a SpectraMax L that injected 50μl of the Substrate Reagent and measured luminescence for a period of 1minute.

Analysis:

The maximum luminescence value after Substrate Reagent addition fromeach sample was plotted. MSSA sample RLU values were used to determine aCutoff calculated using the following formula: (Mean MSSA RLU+3*SD MSSARLU).

Results:

FIG. 23 shows the results of S. aureus growth at 4, 8, 16, 32, 64, and128 ug/mL of cefoxitin. FIG. 24 shows the RLU values obtained by theNRTP assay in the presence of 4, 8, 16, 32, 64, and 128 ug/mL cefoxitin.The x-axis in FIG. 24 is set at the MSSA RLU cutoff value.

As can be seen in FIG. 23, MRSA NRS25 exhibited a MIC of 128 ug/mLcefoxitin while MRSA S7 exhibited a MIC of 64 ug/mL cefoxitin.Correspondingly, MRSA NRS25 exhibited appreciable luminescence above theMSSA RLU cutoff to a cefoxitin concentration up to 64 ug/mL cefoxitinwhile MRSA S7 exhibited luminescence above the MSSA RLU cutoff to acefoxitin concentration up to 32 ug/mL.

Based on the above data, the NRTP assay demonstrates that RLU valuesobtained from the assay correlate with MIC results and thus the NRTPassay may be used to develop antibiotic susceptibility assays.

Example 9: Transcript Reporter Assay: Mechanism of Conformational Changeby RBS-Blocking Cis-Repression of Luxab Translation Activated by themecA Gene Transcript of Mrsa

As described above, a reporter transcript can be designed such thattranslation of the reporter gene sequence is blocked by cis-repressionof the ribosome-binding site (RBS) of the reporter gene.

The following tools were used for designing the reporter transcripts ofthe invention.

1) RNA secondary structure was calculated using secondary structureprogram, such as Mfold.

2) Intermolecular RNA interactions were calculated using a softwareprogram such as RNA-RNA InterACTion prediction using Integer Programming(RactIP).

3) RNA secondary structure was visualized using Visualization Applet forRNA (VARNA).

FIG. 25 shows a secondary structure of the mecA transcript generatedbased on the lowest energy conformation calculated by MFold andvisualized with VARNA. The terminal loop 23 (T23) contains a YUNRsequence UUGG consisting of bases 1,487-1,490 of the mecA transcriptsequence. Analysis of the secondary structure of the mecA genetranscript revealed several ssRNA regions that were suitable fordesigning a cis-repressed luxAB reporter that can be de-repressed viainteractions between the reporter and an ssRNA region.

As shown in detail in FIG. 26, the terminal loop 23 (T23) of the mecAtranscript contains a YUNR consensus sequence. A YUNR(pYrimidine-Uracil-Nucleotide-puRine) consensus sequence has been shownto be a critical target for intermolecular RNA complexes in naturalsystems. A cis-repressing sequence was designed to form a stem-loopstructure with the RBS of the reporter sequence, such that thecis-repressing sequence blocks binding of an RNA polymerase to the RBSof the reporter sequence. The reporter sequence was exposed upon bindingof the loop of the cis-repressing stem-loop structure with T23 of themecA transcript.

As shown in FIG. 27, a cis-repressing sequence 2701 was added to the 5′terminus of the luxAB genes and designed to form a stem-loop structurethat blocks the RBS sequence (“AAGGAA”) 2702 of the luxA gene. Thecis-repressing stem-loop structure was predicted to block the luxA RBS(“AAGGAA”) sequence, based on the lowest energy conformation of theluxAB transcript including the cis-repressing sequence at the 5′terminus of the luxAB transcript, as calculated by MFold and visualizedwith VARNA.

The first 61 nucleotides of the cis-repressed luxAB genes are shown inFIG. 7, up to the start codon AUG of the luxA gene. The RBS sequence“AAGGAA” includes bases 47-52. This terminal loop of the reportertranscript was designed to interact with (bind to) the terminal loop 23(T23) of the mecA transcript, which contains a YUNR sequence.

The terminal loop of the cis-repressing sequence was designed tointeract with T23 of the mecA transcript, such that hybridization of thecis-repressed luxAB transcript and the mecA transcript via theinteraction of the loop from the cis-repressing stem-loop structure andT23 of the mecA transcript results in exposure of the RBS of the luxAgene. FIG. 28 shows the predicted inter-molecular interactions betweenthe mecA T23 sequence and the cis-repressing sequence on the luxABtranscript calculated by RactlP and visualized by VARNA. Lines indicatebase pairing between the mecA transcript and the cis-repressed luxABtranscript. The interaction between the two sequences results inexposure of the luxA RBS sequence AAGGAA and thus de-repression of theluxAB reporter.

Example 10: Transcript Reporter Assay: Methods of Detecting TargetTranscripts or Genes Using a mecA—luxAB Reporter System

In another example, a method for detecting a target mecA gene isprovided using a mecA-luxAB reporter system. Here, mecA is the targettranscript, and luxAB is the reporter molecule.

1. Construction of the Reporter Construct

A vector comprising a reporter construct encoding luxAB can beconstructed via standard molecular biological techniques byincorporating the reporter construct into a shuttle vector capable ofpropagating in both E. coli and S. aureus. The vector can contain anorigin of replication that is functional in E. coli and a selectablemarker that is expressed in E. coli and suitable for allowing the growthof E. coli cells transformed with the vector and grown under selectiveconditions. The vector can also contain an origin of replication that isfunctional in S. aureus and a selectable marker that is expressed in S.aureus and suitable for allowing the growth of E. coli cells transformedwith the vector and grown under selective conditions. Propagation of thevector for conducting in vitro manipulations and for verification ofmanipulations can be accomplished via a suitable laboratory cloningstrain of E. coli and the final modified vector can then be introducedinto S. aureus strains.

The reporter construct can be first introduced into a S. aureus cell fortranscribing the construct and producing the reporter transcript.

2. Construction of a Cis-Repressed Reporter Transcript

Methods are provided for constructing a cis-repressed reportertranscript that can bind to a mecA-target transcript. The reportertranscript can be constructed via standard molecular biologicaltechniques. The luxA and luxB genes serve as reporter genes and can bederived from Vibrio harveyi. The genes lack a transcriptional promoter,and each contains its own ribosomal binding site (RBS). When both theluxA and luxB genes are translated in a cell, the luxA and luxB proteinscomplex to form the active luciferase enzyme (LuxAB). See Farinha, M. A.and A. M. Kropinski, Construction of broad-host-range plasmid vectorsfor easy visible selection and analysis of promoters. J. Bacteriol.,1990. 172(6): p. 3496-3499.

The cis-repressing sequence can be situated upstream of the luxAB genesand downstream of a promoter and includes a sequence that iscomplementary to the luxA RBS. A linker sequence can separate thecomplementary regions of the cis-repressing sequence and the luxAsequence. After transcription of the vector, the complementary regionsof the cis-repressing sequence and the luxA RBS sequence complex,creating a stem loop that prevents docking of a ribosome and hencetranslation.

The stem loop of the reporter transcript is designed to destabilize andform an open complex when it interacts with a naturally-occurring mecAtranscript sequence (endogenous to the cell). To activate translation ofthe luxA gene sequence, the natural mecA transcript serves as atrans-activating RNA that binds to the cis-repressed reporter transcriptand opens the inhibitory stem loop that sequesters the RBS of the luxAgene. Once the RBS is not sequestered by the cis-repressing sequence,translation of luxA can occur. Transcription of the reporter constructis accomplished via operatively linking the reporter sequence to aconstitutive promoter, upstream of the cis-repressing sequence.

An example of a target mecA gene sequence is shown in FIG. 29. Thesequence is a mecA gene loci DNA sequence (from Staphylococcus aureussubsp. aureus SA40, complete genome GenBank: CP003604.1; SEQ ID NO:15)and can be used for generating a reporter construct comprising areporter sequence and a cis-repressing sequence. The −10 position 2901,the transcription start position 2902, the RBS 2903, the coding region(in grey 904) and the transcription termination sequence 2905 are shown.

FIG. 30 shows an exemplary mecA transcript sequence that can be used fordesigning a reporter transcript (SEQ ID NO:16), according to anembodiment of the invention. The RBS 3001 and the coding sequence 3002are shown for mecA.

FIG. 31 is an example of a luxAB gene loci DNA sequence that can be usedfor designing a reporter transcript, according to an embodiment of theinvention. The luxAB gene loci DNA sequence was obtained from Vibriofischeri genes luxA and luxB for luciferase alpha and beta subunits(GenBank: X06758.1) (SEQ ID NO: 17). The −10 position 3101, thetranscription start position 3102, the RBS for lux A 3103, the luxAcoding sequence 3104 (gray shading), the RBS for luxB 3105, and the luxBcoding sequence (gray shading) 3106 are shown.

FIG. 32 is an example of a luxAB transcript sequence that can be usedfor designing a reporter transcript (SEQ ID NO:18). The RBS for lux A3201, the luxA coding sequence 3202 (gray shading), the RBS for luxB3203, and the luxB coding sequence (gray shading) 3204 are shown.

FIG. 33 is an example of a luxAB cis-repressed transcript sequence thatcan be used in a reporter transcript (SEQ ID NO:19). The cis-repressingsequence (dotted line box) 3301, the RBS for lux A 3302, the luxA codingsequence 3303 (gray shading), the RBS for luxB 3304, and the luxB codingsequence (gray shading) 3305 are shown.

3. Methods for Detecting the Presence or Absence of a mecA TargetTranscript Using the Reporter Transcript

Examples are provided for detecting the presence or absence of a mecAtarget transcript in a cell using the reporter transcripts of theinvention. FIG. 34 shows an example of a cell comprising a vector 3400that encodes a reporter transcript 1410, where there is no endogenousmecA transcript in the cell 3401 (e.g., the cell's genome does notcontain the mecA gene). In this case, the cis-repressing sequence 3420binds to the RBS 3430 of the luxAB genes. In some embodiments, thecis-repressing sequence 3420 can bind to a portion of or all of the RBSof the luxA gene, the RBS of the luxB gene, or both. This binding eventblocks and prevents the translation of the luxAB genes, and the reportermolecule (e.g., luciferase) is not produced in the cell. Thus, no signalis detected, indicating the absence of the mecA gene in the cell.

In another example, the cell includes an endogenous mecA transcript(e.g., the cell's genome contains the mecA gene). FIG. 35 shows a vector3400 introduced into a cell 3401. The vector 3400 encodes the reportertranscript 3410, which includes a cis-repressing sequence 3420 and areporter sequence (luxA and luxB genes). When the mecA transcript 3510present in the cell binds to the cis-repressing sequence 1420, theinhibitory hairpin loop opens up and the RBS 3430 for the luxA gene isexposed. Translation of the reporter sequences (luxA and luxB) canoccur, resulting in the formation of a luxAB enzyme 3520. The luxABenzyme 3520 produces a detectable luminescent signal 3530. In thismanner, the transcript reporter vector 3400 reports the presence ofendogenous mecA transcripts 3510 within a cell 3401.

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

REFERENCES CITED

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What is claimed:
 1. A bacterial cell packaging system for packaging areporter nucleic acid molecule into a non-replicative transductionparticle (NRTP) for introduction into a cell, the packaging systemcomprising: a host bacteria cell; a first nucleic acid construct insidethe host bacteria cell, comprising of a bacteriophage genome having afirst bacteriophage gene that contains a non-functional packaginginitiation site sequence, wherein the non-functional packaginginitiation site sequence prevents packaging of the bacteriophage genomeinto the NRTP; and a second nucleic acid construct inside the hostbacteria cell and separate from the first nucleic acid construct,comprising of the reporter nucleic acid molecule having a reporter geneand a second bacteriophage gene that contains a functional packaginginitiation site sequence for facilitating packaging of a replicon of thereporter nucleic acid molecule into the NRTP, wherein the functionalpackaging initiation site sequence on the second nucleic acid constructcomplements the non-functional packaging initiation site sequence in thebacteriophage genome on the first nucleic acid construct.
 2. Thebacterial cell packaging system of claim 1, wherein the reporter gene isluxAB.
 3. The bacterial cell packaging system of claim 1, wherein thereporter gene is operatively linked to a constitutive promoter.
 4. Thebacterial cell packaging system of claim 3, wherein the constitutivepromoter is a Staphylococcus aureus (S. aureus) clpB promoter.
 5. Thebacterial cell packaging system of claim 4, wherein the S. aureus clpBpromoter comprises the sequence of SEQ ID NO:
 12. 6. The bacterial cellpackaging system of claim 1, wherein the bacteriophage genome is a ϕ80αgenome.
 7. The bacterial cell packaging system of claim 1, wherein thefirst bacteriophage gene and the second bacteriophage gene is a ϕ80αsmall terminase (terS) gene.
 8. The bacterial cell packaging system ofclaim 1, wherein the second bacteriophage gene comprises the sequence ofSEQ ID NO:
 9. 9. The bacterial cell packaging system of claim 1, whereinthe bacteriophage genome is an Enterobacteriaceae bacteriophage P1genome.
 10. The bacterial cell packaging system of claim 1, wherein thefirst bacteriophage gene and the second bacteriophage gene is a pacAterminase gene of Enterobacteriaceae bacteriophage P1.
 11. The bacterialcell packaging system of claim 1, wherein the second bacteriophage genecomprises the sequence of SEQ ID NO:
 7. 12. The bacterial cell packagingsystem of claim 1, wherein the reporter nucleic acid molecule is aplasmid comprising at least one origin of replication.
 13. The bacterialcell packaging system of claim 12, wherein the at least one origin ofreplication is a pT181 origin of replication.
 14. The bacterial cellpackaging system of claim 12, wherein the at least one origin ofreplication comprises the sequence of SEQ ID NO:
 5. 15. The bacterialcell packaging system of claim 1, wherein the reporter nucleic acidmolecule is a plasmid comprising the sequence of SEQ ID NO:
 14. 16. Abacterial cell packaging system for packaging a reporter plasmid into anon-replicative transduction particle (NRTP) for introduction into acell, the packaging system comprising: a host bacteria cell; a firstnucleic acid construct inside the host bacteria cell, comprising of aϕ80α bacteriophage genome having a non-functional packaging initiationsite sequence within a ϕ80α small terminase (terS) gene, wherein thenon-functional packaging initiation site sequence prevents packaging ofthe ϕ80α bacteriophage genome into the NRTP; and a second nucleic acidconstruct inside the host bacteria cell and separate from the firstnucleic acid construct, comprising of the reporter plasmid having aluxAB reporter gene operatively linked to a S. aureus clpB promotercomprising the sequence of SEQ ID NO: 12, a pT181 origin of replicationcomprising the sequence of SEQ ID NO: 5, and a ϕ80α small terminase(terS) gene comprising the sequence of SEQ ID NO: 9 that contains afunctional packaging initiation site sequence that facilitates packagingof a replicon of the reporter plasmid into the NRTP, wherein thefunctional packaging initiation site sequence in the ϕ80α smallterminase (terS) gene on the second nucleic acid construct complementsthe non-functional packaging initiation site sequence in the ϕ80αbacteriophage genome on the first nucleic acid construct.
 17. Abacterial cell packaging system for packaging a reporter plasmid into anon-replicative transduction particle (NRTP) for introduction into acell, the packaging system comprising: a host bacteria cell; a firstnucleic acid construct inside the host bacteria cell, comprising of anEnterobacteriaceae bacteriophage P1 genome having a non-functionalpackaging initiation site sequence within a bacteriophage P1 pacAterminase gene, wherein the non-functional packaging initiation sitesequence prevents packaging of the bacteriophage P1 genome into theNRTP; and a second nucleic acid construct inside the host bacteria celland separate from the first nucleic acid construct, comprising of thereporter plasmid having a luxAB reporter gene operatively linked to anEnterobacteriaceae β-lactamase gene bla promoter, a pBBR1-derived originof replication, and a bacteriophage P1 pacA terminase gene comprisingthe sequence of SEQ ID NO: 7 that contains a functional packaginginitiation site sequence that facilitates packaging of a replicon of thereporter plasmid into the NRTP, wherein the functional packaginginitiation site sequence in the bacteriophage P1 pacA terminase gene onthe second nucleic acid construct complements the non-functionalpackaging initiation site sequence in the bacteriophage P1 genome on thefirst nucleic acid construct.